Tissue plasminogen activator-like protease

ABSTRACT

The present invention relates to a novel t-PALP protein which is a member of the serine protease family. In particular, isolated nucleic acid molecules are provided encoding the human t-PALP protein. t-PALP polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of t-PALP activity. Also provided are diagnostic methods for detecting circulatory system-related disorders and therapeutic methods for treating circulatory system-related disorders.

This application is a divisional of U.S. application Ser. No.10/057,951, filed Jan. 29, 2002, now U.S. Pat. No. 6,815,534 which is adivisional of U.S. application Ser. No. 09/411,977, filed Oct. 4, 1999,now U.S. Pat. No. 6,372,473, which is a continuation-in-part of U.S.application Ser. No. 09/084,491, filed May 27, 1998, now abandoned whichclaims benefit under 35 U.S.C. § 119(e) of U.S. Provisional ApplicationNo. 60/048,000, filed on May 28, 1997, each of which, including itscorresponding sequence listing(s), is hereby incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present invention relates to a novel human gene encoding apolypeptide which is a homolog of tissue-type plasminogen activator(t-PA). More specifically, isolated nucleic acid molecules are providedencoding a human polypeptide named tissue-plasminogen activator-likeprotease, hereinafter referred to as “t-PALP”. t-PALP polypeptides arealso provided, as are vectors, host cells and recombinant methods forproducing the same. Also provided are diagnostic methods for detectingdisorders related to the circulatory system and therapeutic methods fortreating such disorders. The invention further relates to screeningmethods for identifying agonists and antagonists of t-PALP activity.

BACKGROUND OF THE INVENTION

The plasmin coagulation system is activated in response to vascularinjury. Within a few minutes of the injury, prothrombin is activatedthrough the coagulation cascade to give rise to thrombin. Thrombin thenconverts fibrinogen to insoluble fibrin, which then interdigitates withand strengthens the primary platelet. Abnormal blood clotting can leadto many vascular diseases, such as stroke, deep-vein thrombosis,peripheral arterial occlusion, pulmonary embolism, andmyocardiothrombosis, each of which constitutes a major health risk. Suchdiseases are primarily caused by partial or total occlusion of a bloodvessel by a blood clot. Such clots consist essentially of a mass offibrin and platelets. The prevention of clot formation and thedissolution of existing clots are two major therapeutic avenuesfrequently used for the treatment of disease states related to bloodclots. Prevention of clot formation is primarily achieved through theinhibition of thrombin activity, whereas the dissolution of existingclots is frequently achieved by the activation of plasminogen whichdissolves the existing blood clot (thereby affecting the fibrinolysispathway).

The fibrinolytic system is activated by the deposition of fibrin. Theconversion of fibrinogen to fibrin results in the exposure of manylysine residues on the surface of the molecule. A factor released fromendothelial cells, termed tissue-type plasminogen activator (t-PA),activates plasminogen. Only upon activation can plasminogen bind toexposed lysine residues on the surface of fibrin, resulting in thedegradation of fibrin, and, ultimately, the degradation of the bloodclot itself.

In man and other animals, t-PA plays an essential role in thedissolution of fibrin clots (see, e.g., Verstraete and Collen, (1986)Blood 67:1425). t-PA is composed of several domains which share sequencehomology with other proteins. These are the fibronectin finger-likedomain, the epidermal growth factor-like domain, the kringle domain (ofwhich t-PA has two), and the protease domain (Pennica, D., et al.,(1983) Nature 301:214–221; Banyai, L., et al., (1983) FEBS Lett.163:37–41). Only the function of the protease domain (residues 276–527)has been unambiguously defined. This finding was first based on theobserved sequence homology with other known serine proteases. Morerecently, limited reduction of the two-chain form of t-PA has allowedthe direct isolation and functional characterization of the proteaseregion (Rijken and Groeneveld, (1986) J. Biol. Chem., 261:3098).

In addition to the role played by human t-PA and related protease-likemolecules in the fibrinolytic system, this same family of molecules alsoplay important roles in carcinogenesis. On the one hand, numerousstudies have implicated the plasminogen activator and/or proteaseactivity of t-PA and related molecules in promoting progression ofcarcinogenesis and metastasis (for example see: Alizadeh, H., (1995)Curr. Eye Res. 14:449; Yamashita, J., (1993) Br. J. Cancer 68:524;Yamashita, J., (1992) Int. J. Clin. Lab Res. 21:227; Koller, A., (1984)Eur. Urol. 10:389). As such, inhibitors of the plasminogen activatorand/or protease activity of t-PA and related molecules may provideuseful therapeutics in combating cancer.

On the other hand, there is also now a large body of evidence whichshows that specific domains from proteins such as t-PA can actuallyinhibit tumorigenesis and metastasis by inhibiting endothelialcell-mediated vascularization (i.e. angiogenesis) which is required fortumor growth. The specific domains mediating such anti-angiogenicactivity have been identified as “kringle” domains. Kringle domains aretriple-looped, disulfide cross-linked domains occurring with varyingcopy numbers in some serine proteases and plasma proteins. The kringledomain has been found in proteins such as: Apolipoprotein A (38 copies);Blood coagulation factor XII (Hageman factor) (1 copy); Hepatocytegrowth factor (HGF) (4 copies); Hepatocyte growth factor-like protein (4copies); Hepatocyte growth factor activator (1 copy); Plasminogen (5copies); Thrombin (2 copies); Urokinase-type plasminogen activator (1copy); and Tissue plasminogen activator (TPA) (2 copies). The signaturepattern of a kringle domain is [F/Y]-C-R-N-P-[D/N/R] (SEQ ID NO:28),where C (cysteine) is involved in disulfide bond formation.

Kringle domains appear to be effective inhibitors of endothelial cellangiogenesis, and thus, effective inhibitors of tumorigenesis andmetastasis. It has been demonstrated, for example, that a four-kringledomain containing protein called HGF/NK4 inhibits invasion of multipletumorigenic cell types in both in vitro and in vivo assays (Date, K., etal. (1998) Oncogene 17:3045). Similarly, angiostatin (a fragment ofplasminogen containing four kringle domains) has also been shown toinhibit tumor vascularization, growth, and metastasis (O'Reilly M. S.,et al., (1994) Cell 79:315; O'Reilly, M. S., et al., (1996) Nat. Med.2:689). Furthermore, a fragment of plasminogen containing just threekringle domains has been demonstrated to markedly reduce growth ofmalignant brain tumors in mice (Joe, J. Y., et al., (1999) Int. J.Cancer 82:694). Finally, it has also been demonstrated that a singlekringle domain of angiostatin is sufficient to significantly inhibitendothelial cell angiogenesis (Cao, Y., et al., (1996) J. Biol. Chem.271:29461). Therefore, t-PALP polynucleotides and/or polypeptides of theinvention may provide particularly good therapeutic molecules for use intreating cancer and/or tumorigenesis, as well as in therapeuticallymodulating angiogenesis.

There is a clear need, therefore, for identification andcharacterization for such enzymes that influence the fibrinolyticsystem, both normally and in disease states. In particular, there is aneed to isolate and characterize additional human tissue plasminogenactivator and related protease-like molecules which possess suchfunctions as the activation of plasminogen and may be employed,therefore, for preventing, ameliorating or correcting dysfunctions ordisease states or, alternatively, augmenting the positive, naturalactions of such enzymes.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of the t-PALPpolypeptide having the complete amino acid sequence shown in SEQ ID NO:2or the complete amino acid sequence encoded by the cDNA clone depositedas plasmid DNA HMSIB42 (ATCC Deposit Number 209023) on May 8, 1997. Thenucleotide sequence determined by sequencing the deposited t-PALP clone,which is shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1), contains an openreading frame encoding a complete polypeptide of 263 amino acidresidues, including an initiation codon encoding an N-terminalmethionine at nucleotide positions 124–126, and a predicted molecularweight of about 28.2 kDa. Nucleic acid molecules of the inventioninclude those encoding the complete amino acid sequence excepting theN-terminal methionine shown in SEQ ID NO:2, or the complete amino acidsequence excepting the N-terminal methionine encoded by the cDNA clonein ATCC Deposit Number 209023, which molecules also can encodeadditional amino acids fused to the N-terminus of the t-PALP amino acidsequence.

The t-PALP protein of the present invention shares sequence homologywith the translation product of the human mRNA for t-PA (FIG. 2) (SEQ IDNO:3), including the following conserved domains: (a) the predictedkringle domain of about 60 amino acids and (b) the predicted proteasedomain of about 179 amino acids. t-PA is thought to be important in theregulation of blood clotting and disorders related thereto. The homologybetween t-PA and t-PALP indicates that t-PALP may also be involved inthe regulation of normal and abnormal clotting in such conditionsincluding many vascular diseases, such as stroke, deep-vein thrombosis,peripheral arterial occlusion, pulmonary embolism, andmyocardiothrombosis.

The encoded polypeptide has a predicted leader sequence of about 21amino acids underlined in FIGS. 1A, 1B, and 1C. The amino acid sequenceof the predicted mature t-PALP protein is also shown in FIGS. 1A, 1B,and 1C, as amino acid residues 22–263 and as residues 1–242 in SEQ IDNO:2.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding a full-length t-PALP polypeptide having the complete amino acidsequence in SEQ ID NO:2 excepting the N-terminal methionine (i.e.,positions −20 to 242 of SEQ ID NO:2) or the complete amino acid sequenceexcepting the N-terminal methionine encoded by the cDNA clone containedin the ATCC Deposit No. 209023; (b) a nucleotide sequence encoding amature t-PALP polypeptide having the amino acid sequence in SEQ ID NO:2from residue 1 to 242 or as encoded by the cDNA clone contained in theATCC Deposit No. 209023; (c) a nucleotide sequence encoding thepredicted kringle domain of the t-PALP polypeptide having the amino acidsequence at positions 4 to 63 in SEQ ID NO:2 or as encoded by the cDNAclone contained in the ATCC Deposit No. 209023; (d) a nucleotidesequence encoding a polypeptide comprising the predicted protease domainof the t-PALP polypeptide having the amino acid sequence at positions 64to 242 in SEQ ID NO:2 or as encoded by the cDNA clone contained in theATCC Deposit No. 209023; and (e) a nucleotide sequence complementary toany of the nucleotide sequences in (a), (b), (c) or (d) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical (or 10% different), and more preferably at least95%, 96%, 97%, 98% or 99% identical (or 5%, 4%, 3%, 2% or 1% differentfrom), to any of the nucleotide sequences in (a), (b), (c), (d) or (e)above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide in (a), (b), (c), (d) or(e) above. This polynucleotide which hybridizes does not hybridize understringent hybridization conditions to a polynucleotide having anucleotide sequence consisting of only A residues or of only T residues.An additional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of a t-PALPpolypeptide having an amino acid sequence in (a), (b), (c) or (d) above.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production oft-PALP polypeptides or peptides by recombinant techniques.

The invention further provides an isolated t-PALP polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) theamino acid sequence of the full-length t-PALP polypeptide having thecomplete amino acid sequence shown in SEQ ID NO:2 excepting theN-terminal methionine (i.e., positions −20 to 242 of SEQ ID NO:2) or thecomplete amino acid sequence excepting the N-terminal methionine encodedby the cDNA clone contained in the ATCC Deposit No. 209023; (b) theamino acid sequence comprising the mature form of the t-PALP polypeptidehaving the amino acid sequence at positions 1 to 242 in SEQ ID NO:2 oras encoded by the cDNA clone contained in the ATCC Deposit No. 209023;(c) the amino acid sequence comprising the predicted kringle domain ofthe t-PALP polypeptide having the amino acid sequence at positions 4 to63 in SEQ ID NO:2 or as encoded by the cDNA clone contained in the ATCCDeposit No. 209023; and (d) the amino acid sequence comprising thepredicted protease domain of the t-PALP polypeptide having the aminoacid sequence at positions 64 to 242 in SEQ ID NO:2 or as encoded by thecDNA clone contained in the ATCC Deposit No. 209023. The polypeptides ofthe present invention also include polypeptides having an amino acidsequence at least 80% identical (that is, 20% different), morepreferably at least 90% identical (10% different), and still morepreferably 95%, 96%, 97%, 98% or 99% identical to (which also may beexpressed as 5%, 4%, 3%, 2% or 1% different from) those described in(a), (b), (c) or (d) above, as well as polypeptides having an amino acidsequence with at least 90% similarity, and more preferably at least 95%similarity, to those above.

An additional embodiment of this aspect of the invention relates to apeptide or polypeptide which comprises the amino acid sequence of anepitope-bearing portion of a t-PALP polypeptide having an amino acidsequence described in (a), (b) or (c) above. Peptides or polypeptideshaving the amino acid sequence of an epitope-bearing portion of a t-PALPpolypeptide of the invention include portions of such polypeptides withat least six or seven, preferably at least nine, and more preferably atleast about 30 amino acids to about 50 amino acids, althoughepitope-bearing polypeptides of any length up to and including theentire amino acid sequence of a polypeptide of the invention describedabove also are included in the invention.

In another embodiment, the invention provides an isolated antibody thatbinds specifically to a t-PALP polypeptide having an amino acid sequencedescribed in (a), (b), (c) or (d) above. The invention further providesmethods for isolating antibodies that bind specifically to a t-PALPpolypeptide having an amino acid sequence as described herein. Suchantibodies are useful diagnostically or therapeutically as describedbelow.

The invention also provides for pharmaceutical compositions comprisingt-PALP polypeptides, particularly human t-PALP polypeptides, which maybe employed, for instance, to treat many vascular diseases, such asstroke, deep-vein thrombosis, peripheral arterial occlusion, pulmonaryembolism, and myocardiothrombosis. Further uses of t-PALP may includeinduction of growth of hepatocytes and regeneration of liver tissue.Methods of treating individuals in need of t-PALP polypeptides are alsoprovided.

The invention further provides compositions comprising a t-PALPpolynucleotide or an t-PALP polypeptide for administration to cells invitro, to cells ex vivo and to cells in vivo, or to a multicellularorganism. In certain particularly preferred embodiments of this aspectof the invention, the compositions comprise a t-PALP polynucleotide forexpression of a t-PALP polypeptide in a host organism for treatment ofdisease. Particularly preferred in this regard is expression in a humanpatient for treatment of a dysfunction associated with aberrantendogenous activity of a t-PALP

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a biological activity ofthe t-PALP polypeptide, which involves contacting an enzyme which isactivated by the t-PALP polypeptide with the candidate compound in thepresence of a t-PALP polypeptide, assaying proteolytic activity of theplasminogen-like molecule in the presence of the candidate compound andof t-PALP polypeptide, and comparing the plasminogen-like moleculeactivity to a standard level of activity, the standard being assayedwhen contact is made between the plasminogen-like molecule and in thepresence of the t-PALP polypeptide and the absence of the candidatecompound In this assay, an increase in plasminogen-like moleculeactivity over the standard indicates that the candidate compound is anagonist of t-PALP activity and a decrease in plasminogen-like moleculeactivity compared to the standard indicates that the compound is anantagonist of t-PALP activity.

In another aspect, a screening assay for agonists and antagonists isprovided which involves determining the effect a candidate compound hason t-PALP binding to a plasminogen-like molecule. In particular, themethod involves contacting the plasminogen-like molecule with a t-PALPpolypeptide and a candidate compound and determining whether t-PALPpolypeptide binding to the plasminogen-like molecule is increased ordecreased due to the presence of the candidate compound. In this assay,an increase in binding of t-PALP over the standard binding indicatesthat the candidate compound is an agonist of t-PALP binding activity anda decrease in t-PALP binding compared to the standard indicates that thecompound is an antagonist of t-PALP binding activity.

It has been discovered that t-PALP is expressed not only in activatedmonocytes, but in a number of other cells and tissues includingcerebellum, smooth muscle, resting and PHA-treated T-cells,GM-CSF-treated macrophages, frontal cortex of the brain, breast lymphnode, chronic lymphocytic leukemic spleen, and several others.Therefore, nucleic acids of the invention are useful as hybridizationprobes for differential identification of the tissue(s) or cell type(s)present in a biological sample. Similarly, polypeptides and antibodiesdirected to those polypeptides are useful to provide immunologicalprobes for differential identification of the tissue(s) or cell type(s).In addition, for a number of disorders of the above tissues or cells,particularly of the circulatory system, significantly higher or lowerlevels of t-PALP gene expression may be detected in certain tissues(e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum,plasma, urine, synovial fluid or spinal fluid) taken from an individualhaving such a disorder, relative to a “standard” t-PALP gene expressionlevel, i.e., the t-PALP expression level in healthy tissue from anindividual not having the circulatory system disorder. Thus, theinvention provides a diagnostic method useful during diagnosis of such adisorder, which involves: (a) assaying t-PALP gene expression level incells or body fluid of an individual; (b) comparing the t-PALP geneexpression level with a standard t-PALP gene expression level, wherebyan increase or decrease in the assayed t-PALP gene expression levelcompared to the standard expression level is indicative of disorder inthe circulatory system.

A further aspect of the invention is related to the relativeclot-specificities which t-PALP and t-PA may possess. For example,t-PALP may have a higher or lower affinity for exerting its proteolyticactivity with respect to a blood clot which localized itself to thelungs than does t-PA. In addition, t-PALP may have a higher or loweraffinity for a specific constituent of a given blood clot than doest-PA. Thus, the t-PALP molecule may prove useful as an agent which,directly or indirectly, results in the dissolution of a blood clot witha higher or lower activity than other agents.

An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of t-PALP activityin the body comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an isolated t-PALPpolypeptide of the invention or an agonist thereof.

A still further aspect of the invention is related to a method fortreating an individual in need of a decreased level of t-PALP activityin the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of an t-PALPantagonist. Preferred antagonists for use in the present invention aret-PALP-specific antibodies.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, and 1C show the nucleotide sequence (SEQ ID NO:1) anddeduced amino acid sequence (SEQ ID NO:2) of t-PALP.

The predicted leader sequence of about 21 amino acids is underlined.Note that the methionine residue at the beginning of the leader sequencein FIGS. 1A, 1B, and 1C is shown in position number (positive) 1,whereas the leader positions in the corresponding sequence of SEQ IDNO:2 are designated with negative position numbers. Thus, the leadersequence positions 1 to 21 in FIGS. 1A, 1B, and 1C correspond topositions −21 to −1 in SEQ ID NO:2.

FIG. 2 shows the regions of identity between the amino acid sequences ofthe t-PALP protein and amino acid residues 191 to 516 of the translationproduct of the human mRNA for t-PA (residues 1–325 in SEQ ID NO:3),determined by the computer program Bestfit (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711) using the defaultparameters. Conserved regions of identity include from Ser-12 to Gly-21,from Ser-22 to Thr-38, from Ser-39 to Trp-49, from Leu-50 to Ser-62,from Gly-63 to Val-84, from Ser-85 to Glu-97, from Arg-100 to Glu-118,from Ala-119 to Glu-127, from Val-128 to Ala-143, from Val-146 toLys-163, from Lys-164 to Ile-180, from Ala-186 to Leu-200, from Lys-201to Leu-220, from Ser-221 to Val-236, from Val-237 to Gln-248, and fromGlu-249 to Ala-263 of SEQ ID NO:2. Polynucleotides encoding each ofthese conserved domains are also encompassed by the invention, as wellas combinations of the conserved domains. These conserved domains arepreferred embodiments of the present invention.

FIG. 3 shows an analysis of the t-PALP amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity and hydrophobicity; amphipathicregions; flexible regions; antigenic index and surface probability areshown, as predicted for the amino acid sequence of SEQ ID NO:2 using thedefault parameters of the recited computer programs. In the “AntigenicIndex—Jameson-Wolf” graph, the positive peaks indicate locations of thehighly antigenic regions of the t-PALP protein, i.e., regions from whichepitope-bearing peptides of the invention can be obtained. Antigenicpolypeptides include from about Ala-19 to about Gly-24, from aboutAsn-29 to about Cys-46, from about Ala-52 to about Ala-57, from aboutVal-61 to about Asn-66, from about Ser-68 to about Trp-81, from aboutSer-85 to about Gln-107, from about Glu-115 to about Gln-129, from aboutPro-138 to about Ala-145, from about Gln-154 to about Gly-167, fromabout Tyr-192 to about Arg-215, from about Thr-224 to about Val-236,from about Thr-240 to about Thr-252, and from about Ala-258 to aboutAla-263 of the amino acid sequence of SEQ ID NO:2 using the numberingscheme of FIGS. 1A, 1B, and 1C. Polynucleotides encoding these antigenicpolypeptides are also encompassed by the invention. These antigenicpolypeptides are preferred embodiments of the present invention.

The data presented in FIG. 3 are also represented in tabular form inTable 1 (below). The columns are labeled with the headings “Residue”,“Pos. #”, and Roman Numerals I–XIV. The column headings refer to thefollowing features of the amino acid sequence in SEQ ID NO:2 using thenumbering scheme of FIGS. 1A, 1B, and 1C: “Residue”: amino acid residueof t-PALP as shown in and FIGS. 1A, 1B, and 1C; “Pos. #”: position ofthe corresponding residue within SEQ ID NO:2 using the numbering schemeof FIGS. 1A, 1B, and 1C; I: Alpha, Regions—Garnier-Robson; II: Alpha,Regions—Chou-Fasman; III: Beta, Regions—Garnier-Robson; IV: Beta,Regions—Chou-Fasman; V: Turn, Regions—Garnier-Robson; VI: Turn,Regions—Chou-Fasman; VII: Coil, Regions—Gamier-Robson; VIII:Hydrophilicity Plot—Kyte-Doolittle; IX: Hydrophobicity Plot—Hopp-Woods;X: Alpha, Amphipathic Regions—Eisenberg; XI: Beta, AmphipathicRegions—Eisenberg; XII: Flexible Regions—Karplus-Schulz; XIII: AntigenicIndex—Jameson-Wolf; and XIV: Surface Probability Plot—Emini.

FIG. 4 shows an analysis of tumor growth of TSU cells transfected witht-PALP as determined by a chick chorioallantoic membrane (CAM) assay.Using a protocol based on that of Brooks, et al. (See, Cell 79:1157–64(1994); See also, Brooks, et al., Cell 92:391–400 (1998)), the effectsof t-PALP on the growth of TSU cells were analyzed in a CAM assay.Fifteen to twenty eggs were used for each treatment and the mean +/−standard error of tumor mass (mg/CAM) was calculated. The resulting datawere subjected to the student's t-test for statistical analysis. Thefigure shows tumor weight (in mg per CAM) plotted against either tumorcells transfected with expression vector (labeled as “TSU-pCDNA3”) onlyor against tumor cells transfected with a t-PALP expression vector(labeled as “TSU-t-PALP”).

FIG. 5 shows the effect of conditioned medium from TSU cells transientlytransfected with a t-PALP expression construct. Cell number was used toassess the effect of t-PALP on endothelial cells. Aliquots of either 50,100 or 200 microliters of conditioned medium from the transientlytransfected TSU cell cultures was added to the culture medium ofendothelial cells. Treated cultures were then incubated at 37° C. Thenumber of cells in each culture was then determined.

Results of the experiments are plotted in FIG. 5 as cell number (×200)against 50, 100 or 200 microliters of either the control conditionedmedium (labeled as “Control CM”) or conditioned medium fromt-PALP-transfected TSU cells (labeled as “t-PALP CM”). The data areplotted as the mean +/− standard deviation. Significance according tothe student's t-test is indicated by an asterisk.

FIG. 6 shows that tumor growth in nude mice is significantly inhibitedwhen the mice are injected with tumorigenic cells transientlytransfected with t-PALP cDNA as compared to tumorigenic cellstransiently transfected with expression vector only. In this assay,tumorigenic TSU cells were either transfected with expression vectoronly or transfected with t-PALP cDNA. The cells were then harvested withwith 10 mM EDTA-PBS. One million tumor cells suspended in 0.2 ml of DMEMper side were injected subcutaneously into 6 week old nude mice. Fivemice were used in each group. The tumor size was measured twice a week.The mice were sacrificed 4 weeks later and the tumors were removed,weighed, and measured. The mean +/− standard error was calculated andsubjected to student's t-test. Tumor size of mock transfected cells isindicated by closed circles. Tumor size of t-PALP transfected cells isindicated by closed squares.

TABLE 1 Residue Pos. # I II III IV V VI VII VIII IX X XI XII XIII XIVMet 1 A A . . . . . −1.16 0.77 . . . −0.60 0.25 Leu 2 A A . . . . .−1.62 1.26 . . . −0.60 0.21 Leu 3 A A . . . . . −1.23 1.47 . . . −0.600.12 Ala 4 A A . . . . . −1.43 1.44 * . . −0.60 0.21 Trp 5 A A . . . . .−1.74 1.33 * . . −0.60 0.26 Val 6 A A . . . . . −1.96 1.43 . . . −0.600.27 Gln 7 A A . . . . . −2.00 1.43 . . . −0.60 0.22 Ala 8 A A . . . . .−1.49 1.57 . . . −0.60 0.16 Phe 9 A A . . . . . −0.90 1.04 . . . −0.600.28 Leu 10 A A . . . . . −1.21 0.80 . . . −0.60 0.26 Val 11 A A . . . .. −1.17 1.01 . . . −0.60 0.26 Ser 12 A A . . . . . −1.98 1.20 . . .−0.60 0.25 Asn 13 A A . . . . . −1.98 1.10 . . . −0.60 0.25 Met 14 A A .. . . . −1.28 0.91 . . . −0.60 0.34 Leu 15 A A . . . . . −1.06 0.27 . .. −0.30 0.43 Leu 16 A A . . . . . −0.44 0.39 . . . −0.30 0.27 Ala 17 A A. . . . . −0.49 0.74 . . . −0.60 0.43 Glu 18 A A . . . . . −0.79 0.56 .. . −0.53 0.52 Ala 19 A A . . . . . −0.53 0.26 . . . −0.16 0.84 Tyr 20 .A . . T . . −0.07 0.00 . . . 0.31 0.82 Gly 21 . . . . T T . 0.08 −0.07 .. F 1.53 0.47 Ser 22 . . . . T T . −0.03 0.50 . . F 0.70 0.25 Gly 23 . .. . T T . −0.32 0.79 * . F 0.63 0.14 Gly 24 . . . . T T . 0.27 0.94 * .F 0.56 0.15 Cys 25 . . B . . . . 0.51 0.51 * . . −0.26 0.18 Phe 26 . . B. . . . 0.51 0.53 . . . −0.33 0.30 Trp 27 . . B . . T . 0.78 0.53 . . .−0.20 0.30 Asp 28 . . B . . T . 0.31 0.60 . . F −0.05 0.75 Asn 29 . . .. T T . 0.41 0.71 . . F 0.35 0.72 Gly 30 . . . . T T . 1.19 0.69 * . F0.50 1.07 His 31 . A . . . . C 1.89 −0.23 . . . 0.65 1.26 Leu 32 . A . .. . C 2.18 −0.23 . . . 0.65 1.35 Tyr 33 . A . . . . C 2.18 −0.63 . . .1.29 2.28 Arg 34 . A B . . . . 1.87 −0.66 . . F 1.58 2.91 Glu 35 . A B .. . . 1.91 −0.67 * . F 1.92 5.09 Asp 36 . . . . T T . 1.73 −0.97 * . F3.06 4.35 Gln 37 . . . . T T . 1.96 −1.30 . . F 3.40 3.43 Thr 38 . . . .. T C 1.99 −0.80 . . F 2.86 2.00 Ser 39 . . . . . T C 1.53 −0.37 . . F2.35 1.85 Pro 40 . . . . . . C 0.72 0.06 . * F 1.34 1.06 Ala 41 . . . .. T C 0.83 0.34 . * F 1.18 0.61 Pro 42 . . . . T T . 0.17 −0.14 * * F1.77 0.89 Gly 43 . . . . T T . −0.33 0.04 * . F 1.30 0.31 Leu 44 . . B .. T . −0.03 0.30 * . . 0.62 0.25 Arg 45 . . B . . . . −0.11 0.20 * . .0.29 0.26 Cys 46 . . B . . T . −0.33 0.69 * . . 0.06 0.28 Leu 47 . . B .. T . −0.12 0.94 * * . −0.07 0.28 Asn 48 . . B . . T . −0.37 0.26 * * .0.10 0.24 Trp 49 . . B . . T . 0.44 0.76 * * . −0.20 0.44 Leu 50 . . B .. . . 0.03 0.59 * . . −0.40 0.93 Asp 51 A . . . . . . 0.36 0.29 . . .−0.01 0.78 Ala 52 A . . . . . . 0.36 0.31 . . F 0.23 0.73 Gln 53 . . B .. T . −0.23 0.09 * . F 0.52 0.73 Ser 54 . . . . . T C −0.24 −0.10 . . F1.41 0.44 Gly 55 . . . . . T C −0.02 0.29 . * F 0.90 0.59 Leu 56 . . . .. T C −0.23 0.29 . * F 0.81 0.34 Ala 57 . . B . . . . −0.50 0.31 . . .0.17 0.40 Ser 58 . . B . . . . −0.80 0.57 . . . −0.22 0.30 Ala 59 . . B. . . . −0.84 0.53 . . . −0.31 0.48 Pro 60 . . B . . . . −1.09 0.27 . .. −0.10 0.47 Val 61 . . B . . . . −0.62 0.27 . * . −0.10 0.36 Ser 62 . .B . . . . −0.03 0.31 . * F 0.05 0.35 Gly 63 . . . . T T . 0.23 0.21 . *F 0.65 0.36 Ala 64 . . . . T T . 0.52 0.29 . * F 0.65 0.67 Gly 65 . . .. T T . 0.49 0.03 . . F 0.65 0.67 Asn 66 . . . . T T . 0.68 0.40 . * .0.35 1.05 His 67 . . B . . T . 1.09 0.54 . . . −0.20 0.56 Ser 68 . . B .. T . 1.43 0.04 . . . 0.25 1.11 Tyr 69 . . B . . T . 1.81 0.01 * . .0.59 1.11 Cys 70 . . . . T T . 2.16 0.04 * . . 1.33 1.26 Arg 71 . . . .T . . 2.16 −0.46 * . F 2.22 1.57 Asn 72 . . B . . T . 2.19 −0.84 * . F2.66 1.73 Pro 73 . . . . T T . 2.28 −1.60 * * F 3.40 5.39 Asp 74 . . . .T T . 2.63 −1.74 * * F 3.06 4.26 Glu 75 . . . . T T . 2.96 −1.74 * * F2.93 5.18 Asp 76 . . . . . T C 2.63 −1.71 * * F 2.60 3.32 Pro 77 . . . .T T . 2.34 −1.71 . * F 2.67 3.07 Arg 78 . . . . T T . 1.89 −0.80 * * F2.54 1.87 Gly 79 . . . . . T C 1.64 −0.23 * * F 2.10 0.60 Pro 80 . . . BT . . 0.79 0.53 * * F 0.79 0.61 Trp 81 . . . B T . . 0.49 0.74 . * .0.43 0.23 Cys 82 . . B B . . . 0.36 1.13 . * . −0.18 0.31 Tyr 83 . . B B. . . 0.24 1.13 . * . −0.39 0.20 Val 84 . . B . . T . −0.00 0.70 . . .−0.20 0.33 Ser 85 . . B . . T . −0.13 0.29 . * F 0.25 0.62 Gly 86 . . .. . T C −0.70 0.14 . * F 0.45 0.39 Glu 87 . . . . T T . −0.24 0.03 . * F0.65 0.39 Ala 88 . . . . . . C −0.00 −0.19 . * F 0.85 0.45 Gly 89 . . .. . . C 0.90 −0.57 . * F 1.15 0.79 Val 90 . . . . . . C 1.31 −1.00 . . F1.15 0.91 Pro 91 A . . . . . . 1.44 −1.00 . . F 1.44 1.77 Glu 92 . . . .T . . 0.78 −1.07 * . F 2.18 2.76 Lys 93 A . . . . . . 1.37 −0.93 * . F2.12 1.99 Arg 94 . . B . . T . 1.71 −1.57 * . F 2.66 2.23 Pro 95 . . . .T T . 1.76 −2.00 * * F 3.40 2.15 Cys 96 . . . . T T . 2.08 −1.31 * * F2.91 0.89 Glu 97 A . . . . T . 1.41 −1.31 * * F 2.17 0.89 Asp 98 . . . .T . . 1.16 −0.74 * * F 2.03 0.31 Leu 99 . . . . T . . 1.04 −0.74 * * .1.85 0.89 Arg 100 . . B . . . . 0.94 −1.31 * * . 1.42 0.89 Cys 101 . . B. . T . 1.30 −0.83 * * F 2.08 0.77 Pro 102 . . . . T T . 1.00 −0.34 * *F 2.64 1.34 Glu 103 . . . . T T . 1.00 −0.64 * * F 3.10 0.92 Thr 104 A .. . . T . 1.22 −0.24 * * F 2.24 2.97 Thr 105 A A . . . . . 0.30 −0.31 *. F 1.53 1.94 Ser 106 . A B . . . . 0.76 −0.06 * . F 1.07 0.92 Gln 107 .A B . . . . 0.38 0.37 * . F 0.16 0.99 Ala 108 . A B . . . . −0.32 0.39 *. . −0.30 0.69 Leu 109 . A . . . . C −0.32 0.69 * . . −0.40 0.45 Pro 110. A . . . . C −0.32 0.79 * . . −0.40 0.37 Ala 111 A A . . . . . −0.020.87 * * . −0.60 0.53 Phe 112 A A . . . . . −0.91 0.37 * * . −0.15 1.12Thr 113 A A . . . . . −0.32 0.37 * * F −0.15 0.51 Thr 114 A A . . . . .0.49 0.34 * . F −0.15 0.87 Glu 115 A A . . . . . 0.11 −0.16 * . F 0.601.74 Ile 116 A A . . . . . 0.40 −0.44 * * F 0.60 1.22 Gln 117 A A . . .. . 1.10 −0.54 * * F 0.90 1.13 Glu 118 A A . . . . . 1.07 −1.03 * . F0.90 1.13 Ala 119 A A . . . . . 1.17 −0.60 * . F 1.20 1.60 Ser 120 . . .. . . C 0.82 −0.86 * . F 1.90 1.42 Glu 121 . . . . . . C 1.12 −0.83 . .F 2.05 0.81 Gly 122 . . . . . T C 1.12 −0.33 . . F 2.25 0.81 Pro 123 . .. . . T C 1.12 −0.83 . . F 3.00 1.01 Gly 124 . . . . . T C 0.86 −1.21 .. F 2.70 1.01 Ala 125 A . . . . T . 1.16 −0.57 . * F 2.05 0.76 Asp 126 AA . . . . . 0.30 −0.60 . . F 1.35 0.85 Glu 127 A A . . . . . −0.06−0.39 * . F 0.75 0.64 Val 128 . A B . . . . −0.43 −0.03 * . . 0.30 0.55Gln 129 . A B . . . . −0.30 −0.03 * * . 0.30 0.33 Val 130 . A B . . . .−0.30 0.40 . * . −0.60 0.30 Phe 131 . A B . . . . −0.30 0.90 . * . −0.600.40 Ala 132 A A . . . . . −0.89 0.66 . * . −0.60 0.37 Pro 133 A . . . .T . −0.84 0.76 . * . −0.20 0.51 Ala 134 A . . . . T . −1.06 0.80 . . .−0.20 0.48 Asn 135 A . . . . T . −0.79 0.44 * * . −0.20 0.74 Ala 136 A .. . . T . 0.02 0.44 . . . −0.20 0.48 Leu 137 A . . . . . . 0.31 0.01 . *. −0.10 0.94 Pro 138 A . . . . T . 0.52 −0.10 . * . 0.70 0.78 Ala 139 A. . . . T . 0.52 −0.50 . * F 1.00 1.34 Arg 140 A . . . . T . −0.07 −0.50. * F 1.00 1.64 Ser 141 A . . . . T . −0.07 −0.69 . * F 1.30 1.07 Glu142 A A . . . . . −0.11 −0.61 . * F 0.90 1.07 Ala 143 A A . . . . . 0.10−0.47 * * . 0.30 0.41 Ala 144 A A . . . . . 0.48 −0.07 * * . 0.30 0.53Ala 145 A A . . . . . −0.49 −0.03 * * . 0.30 0.47 Val 146 A A . . . . .−1.08 0.61 . . . −0.60 0.35 Gln 147 . A B . . . . −1.42 0.80 . . . −0.600.24 Pro 148 . A B . . . . −1.72 0.73 . * . −0.60 0.23 Val 149 . . B B .. . −1.43 0.91 * * . −0.60 0.22 Ile 150 . . B B . . . −0.84 0.66 * * .−0.60 0.17 Gly 151 . . B B . . . 0.12 0.66 * * . −0.60 0.19 Ile 152 . .B B . . . −0.73 0.23 * * . −0.30 0.51 Ser 153 . . B B . . . −0.410.23 * * F −0.15 0.54 Gln 154 . . B B . . . −0.16 −0.46 * * F 0.60 1.06Arg 155 . . B B . . . 0.73 −0.27 . * F 0.60 1.50 Val 156 . . B B . . .0.78 −0.56 * * . 0.75 1.80 Arg 157 . . B . . T . 1.71 −0.56 * * . 1.151.39 Met 158 A . . . . T . 2.01 −0.96 . * . 1.15 1.42 Asn 159 A . . . .T . 2.06 −0.96 . * F 1.30 3.32 Ser 160 A . . . . T . 1.99 −1.60 . * F1.30 3.39 Lys 161 A A . . . . . 2.84 −1.60 * * F 0.90 6.84 Glu 162 A A .. . . . 1.92 −2.21 * * F 0.90 7.11 Lys 163 A A . . . . . 2.18 −1.93 . *F 0.90 4.37 Lys 164 A A . . . . . 1.87 −1.89 . . F 0.90 2.16 Asp 165 A A. B . . . 1.36 −1.40 * . F 0.90 1.80 Leu 166 . A B B . . . 0.97 −0.71 *. F 0.75 0.74 Gly 167 . . B B . . . 0.72 −0.29 . . F 0.45 0.37 Thr 168 .. B B . . . −0.18 0.47 * . F −0.45 0.35 Leu 169 . . B B . . . −1.031.11 * . . −0.60 0.31 Gly 170 . . B B . . . −1.38 1.11 . . . −0.60 0.26Tyr 171 . . B B . . . −1.46 1.11 . . . −0.60 0.18 Val 172 . . B B . . .−1.42 1.31 . . . −0.60 0.15 Leu 173 . . B B . . . −1.71 1.11 . . . −0.600.22 Gly 174 . . B B . . . −1.50 1.30 . . . −0.60 0.14 Ile 175 . . B B .. . −2.01 1.16 . . . −0.60 0.19 Thr 176 . . B B . . . −2.66 1.16 . . .−0.60 0.17 Met 177 . . B B . . . −2.69 1.16 . . . −0.60 0.12 Met 178 . .B B . . . −2.77 1.41 . . . −0.60 0.12 Val 179 . . B B . . . −3.01 1.41 .. . −0.60 0.06 Ile 180 . . B B . . . −3.01 1.43 . . . −0.60 0.06 Ile 181. . B B . . . −3.04 1.50 . . . −0.60 0.04 Ile 182 . . B B . . . −3.031.31 . . . −0.60 0.06 Ala 183 . . B B . . . −2.78 1.17 . . . −0.60 0.08Ile 184 A . . B . . . −2.81 0.91 . . . −0.60 0.11 Gly 185 . . B . . T .−2.81 0.91 . . . −0.20 0.11 Ala 186 . . B . . T . −2.73 0.91 . . . −0.200.08 Gly 187 . . B . . T . −2.19 1.10 . . . −0.20 0.09 Ile 188 . . B . .T . −1.84 0.84 . . . −0.20 0.09 Ile 189 . . B B . . . −1.26 1.17 . . .−0.60 0.14 Leu 190 . . B B . . . −1.16 1.06 . . . −0.60 0.19 Gly 191 . .B B . . . −0.52 1.39 * . . −0.26 0.43 Tyr 192 . . B . . T . −0.07 0.70 *. . 0.63 1.23 Ser 193 . . B . . T . 0.48 0.01 . * . 1.27 2.92 Tyr 194 .. . . T T . 1.41 −0.24 * . . 2.61 2.92 Lys 195 . . . . T T . 2.22−0.67 * . F 3.40 3.73 Arg 196 . . . . T . . 1.76 −1.43 . . F 2.86 4.65Gly 197 . . . . T T . 2.04 −1.13 * . F 2.72 2.45 Lys 198 A . . . . T .2.34 −1.89 * . F 1.98 2.45 Asp 199 A . . . . T . 2.59 −1.89 * . F 1.642.16 Leu 200 A . . . . T . 2.51 −1.49 * . F 1.30 3.79 Lys 201 A A . . .. . 2.40 −1.41 * . F 0.90 2.58 Glu 202 A A . . . . . 2.74 −1.41 * * F0.90 2.58 Gln 203 A A . . . . . 2.74 −1.01 * * F 0.90 5.41 His 204 A A .. . . . 1.89 −1.70 . * F 0.90 5.41 Asp 205 A A . . . . . 2.03 −1.06 . .*F 0.90 2.32 Gln 206 A A . . . . . 1.99 −0.49 * * F 0.45 0.72 Lys 207 A A. . . . . 2.10 −0.89 * * F 0.75 0.91 Val 208 A A . . . . . 2.10−1.39 * * F 0.90 1.07 Cys 209 A A . . . . . 1.53 −1.39 * . . 0.75 1.07Glu 210 A A . . . . . 1.53 −1.17 * * . 0.60 0.53 Arg 211 A A . . . . .1.64 −0.77 * * . 0.75 1.24 Glu 212 A A . . . . . 0.71 −1.41 * * F 0.904.52 Met 213 A A . . . . . 1.26 −1.30 * * F 0.90 1.83 Gln 214 A A . . .. . 1.11 −0.81 * * F 0.90 1.35 Arg 215 A A . . . . . 0.90 −0.13 * * .0.30 0.64 Ile 216 . A B . . . . −0.02 0.30 * * . −0.15 1.00 Thr 217 . AB . . . . −0.32 0.37 * * . −0.30 0.48 Leu 218 . A B . . . . −0.310.36 * * . −0.30 0.33 Pro 219 . A B . . . . −1.01 0.86 * * . −0.60 0.47Leu 220 . . B . . . . −1.43 0.96 * * . −0.40 0.28 Ser 221 . . B . . . .−0.54 0.96 * * . −0.40 0.49 Ala 222 . . . . T . . −0.44 0.67 . . . 0.000.51 Phe 223 . . . . T . . 0.06 0.67 . . . 0.00 0.96 Thr 224 . . . . . .C −0.40 0.47 * . F 0.10 1.04 Asn 225 . . . . . T C 0.41 0.66 * * F 0.150.55 Pro 226 . . . . . T C −0.18 0.16 * . F 0.60 1.10 Thr 227 . . . . TT . −0.44 0.06 * . F 0.65 0.54 Cys 228 A . . . . T . 0.26 0.21 * . .0.10 0.25 Glu 229 A . . B . . . 0.57 −0.19 . . . 0.30 0.27 Ile 230 A . .B . . . 0.61 −0.61 . . . 0.60 0.32 Val 231 A . . B . . . 0.51 −1.10 . .. 0.75 1.19 Asp 232 A . . B . . . −0.03 −1.19 . . F 0.75 1.00 Glu 233 A. . B . . . −0.22 −0.54 . . F 0.90 1.05 Lys 234 A . . B . . . −1.08−0.59 . . F 0.90 1.05 Thr 235 A . . B . . . −0.22 −0.59 . . F 0.75 0.47Val 236 A . . B . . . 0.32 −0.09 . . . 0.30 0.37 Val 237 . . B B . . .0.02 0.40 . . . −0.30 0.27 Val 238 . . B B . . . 0.02 0.79 . . . −0.600.25 His 239 . . B . . T . −0.33 0.70 . . . −0.20 0.58 Thr 240 . . B . .T . −0.23 0.54 . . F 0.10 1.12 Ser 241 . . . . T T . −0.23 0.33 * . F0.80 2.33 Gln 242 . . . . . T C 0.62 0.33 * . F 0.60 1.27 Thr 243 . . .. . . C 1.27 −0.17 . * F 1.00 1.47 Pro 244 . . . . . . C 1.30 −0.23 . *F 1.34 1.70 Val 245 . . B . . . . 1.61 −0.21 . . F 1.48 1.70 Asp 246 . .B . . . . 1.57 −0.61 . . F 2.12 2.04 Pro 247 . . B . . T . 1.27 −0.67. * F 2.66 1.30 Gln 248 . . . . T T . 1.27 −0.71 . . F 3.40 2.35 Glu 249. . . . T T . 1.27 −0.87 . . F 3.06 2.03 Gly 250 . . . . T T . 1.31−0.44 . . F 2.42 2.03 Ser 251 . . . . . . C 0.71 −0.19 . . F 1.53 0.97Thr 252 . . B . . . . 0.58 0.03 . . F 0.39 0.55 Pro 253 . . B . . . .0.58 0.46 . * F −0.25 0.55 Leu 254 . . B . . . . −0.01 0.43 . * F −0.250.72 Met 255 . . B . . . . −0.01 0.54 . . . −0.40 0.50 Gly 256 . . B . .. . −0.02 0.49 . . . −0.40 0.32 Gln 257 . . B . . . . 0.08 0.54 . . F−0.25 0.56 Ala 258 . . B . . . . −0.06 0.29 . . F 0.05 0.88 Gly 259 . .. . . . C 0.17 0.10 . . F 0.25 0.88 Thr 260 . . . . . T C 0.38 0.17 * .F 0.45 0.51 Pro 261 . . . . . T C 0.33 0.20 . . . 0.30 0.65 Gly 262 . .. . . T C −0.06 0.13 . . . 0.30 0.84 Ala 263 . . B . . T . 0.14 0.13 . .. 0.10 0.74

DETAILED DESCRIPTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a t-PALP polypeptide having theamino acid sequence shown in SEQ ID NO:2, which was determined bysequencing a cloned cDNA. The nucleotide sequence shown in FIGS. 1A, 1B,and 1C (SEQ ID NO:1) was obtained by sequencing the HMSIB42 clone, whichwas deposited on May 8, 1997 at the American Type Culture Collection,10801 University Drive, Manassas, Va. 20110-2209, and given accessionnumber ATCC 209023. The deposited clone is contained in the pBluescriptSK(−) plasmid (Stratagene, La Jolla, Calif.).

The t-PALP protein of the present invention shares sequence homologywith the translation product of the human mRNA for t-PA (FIG. 2) (SEQ IDNO:3). t-PA is thought to be an important regulator of the dissolutionof fibrin clots in humans and other animals. Abnormal blood clotting canlead to many vascular diseases, such as stroke, deep-vein thrombosis,peripheral arterial occlusion, pulmonary embolism, andmyocardiothrombosis, each of which constitutes a major health risk. Suchdiseases are primarily caused by partial or total occlusion of a bloodvessel by a blood clot. Such clots consist essentially of a mass offibrin and platelets. The dissolution of existing clots is frequentlyachieved by the activation of plasminogen which dissolves the existingblood clot (thereby affecting the fibrinolysis pathway).

The fibrinolytic system is activated by the deposition of fibrin. t-PAactivates plasminogen and, only upon activation, can plasminogen degradefibrin, and, ultimately, degrade the blood clot itself.

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc., FosterCity, Calif.), and all amino acid sequences of polypeptides encoded byDNA molecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule (thesevalues may also be expressed as at most 10% different, more typically atmost about 5% to about 0.1% different from the actual nucleotidesequence of the sequenced DNA molecule). The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

By “nucleotide sequence” of a nucleic acid molecule or polynucleotide isintended, for a DNA molecule or polynucleotide, a sequence ofdeoxyribonucleotides, and for an RNA molecule or polynucleotide, thecorresponding sequence of ribonucleotides (A, G, C and U), where eachthymidine deoxyribonucleotide (T) in the specified deoxyribonucleotidesequence is replaced by the ribonucleotide uridine (U).

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A, 1B, and 1C (SEQ ID NO:1), a nucleic acid molecule of thepresent invention encoding a t-PALP polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 1A, 1B, and 1C (SEQ IDNO:1) was discovered in a cDNA library derived from activated monocytes.

Additional clones of the same gene were also identified in cDNAlibraries from the following tissues: cerebellum, smooth muscle, restingand PHA-treated T-cells, GM-CSF-treated macrophages, frontal cortex ofthe brain, breast lymph node, chronic lymphocytic leukemic spleen, andseveral others. Thus, in one embodiment, polynucleotides, polypeptides,and antibodies of the invention may be used to distinguish betweentissues. In a preferred embodiment, polynucleotides, polypeptides, andantibodies of the invention may be used to distinguish between tissuesrecited in the above paragraph and in tissues not recited in the aboveparagraph.

A Northern blot analysis of the t-PALP clone of FIGS. 1A, 1B, and 1C(SEQ ID NO:1), or the t-PALP clone contained in ATCC Deposit No. 209023,indicated that 2.5 kb t-PALP message is detectable in heart, brain,placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,thymus, prostate, testis, ovary, small intestine, colon, and peripheralblood leukocytes (see Example 4). Thus, in one embodiment,polynucleotides, polypeptides, and antibodies of the invention may beused to distinguish between tissues. In a preferred embodiment,polynucleotides, polypeptides, and antibodies of the invention may beused to distinguish between tissues recited in the above paragraph andin tissues not recited in the above paragraph. In an additionalpreferred embodiment, polynucleotides, polypeptides, and antibodies ofthe invention may be used to distinguish between human and non-humantissues.

The determined nucleotide sequence of the t-PALP cDNA of FIGS. 1A, 1B,and 1C (SEQ ID NO:1) contains an open reading frame encoding a proteinof 263 amino acid residues, with an initiation codon at nucleotidepositions 124–126 of the nucleotide sequence in FIGS. 1A, 1B, and 1C(SEQ ID NO:1), and a deduced molecular weight of about 28.2 kDa. An invitro transcription/translation analysis of the t-PALP clone shown inSEQ ID NO:1, or the t-PALP clone contained in ATCC Deposit No. 209023,resulted in the production of a protein product of about 35 kDa. Theamino acid sequence of the t-PALP protein shown in SEQ ID NO:2 is about21.3% identical to human mRNA for t-PA (FIG. 2; Degen, S. J., Rajput,B., and Reich, E. (1986) J. Biol. Chem. 261:6972–6985; GenBank AccessionNo. K03021).

The open reading frame of the t-PALP gene shares sequence homology withthe translation product of the human mRNA for t-PA (FIG. 2) (SEQ IDNO:3), including the following conserved domains: (a) the predictedkringle domain of about 59 amino acids, and (b) the predicted proteasedomain of about 179 amino acids. t-PA is thought to be important in theregulation of blood clotting and disorders related thereto. The homologybetween t-PA and t-PALP indicates that t-PALP may also be involved inthe regulation of normal and abnormal clotting in such conditionsincluding many vascular diseases, such as stroke, deep-vein thrombosis,peripheral arterial occlusion, pulmonary embolism, andmyocardiothrombosis. In an additional embodiment t-PALP is involved inthe regulation of angiogenesis and thus, may be useful in the treatmentof cancers; for example solid tumors, including prostate, lung, breast,ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid,biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,bladder, thyroid cancer; primary tumors and metastases; melanomas;glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lungcancer; colorectal cancer; advanced malignancies; blood born tumors(such as leukemias); benign tumors, for example hemangiomas, acousticneuromas, neurofibromas, trachomas, and pyogenic granulomas;artheroscleric plaques; ocular angiogenic diseases, for example diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vesselgrowth) of the eye; rheumatoid arthritis; psoriasis; delayed woundhealing; endometriosis; vasculogenesis; granulations; hypertrophic scars(keloids); nonunion fractures; scleroderma; trachoma; vascularadhesions; myocardial angiogenesis; coronary collaterals; cerebralcollaterals; arteriovenous malformations; ischemic limb angiogenesis;Osler-Webber Syndrome; plaque neovascularization; telangiectasia;hemophiliac joints; angiofibroma; fibromuscular dysplasia; woundgranulation; Crohn's disease; and atherosclerosis.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above, the actual complete t-PALPpolypeptide encoded by the deposited cDNA, which comprises about 263amino acids, may be somewhat longer or shorter. More generally, theactual open reading frame may be anywhere in the range of ±20 aminoacids, more likely in the range of ±10 amino acids, of that predictedfrom the methionine codon at the N-terminus shown in FIGS. 1A, 1B, and1C (SEQ ID NO:1). It will further be appreciated that, depending on theanalytical criteria used for identifying various functional domains, theexact “address” of the kringle and protease domains of the t-PALPpolypeptide may differ slightly from the predicted positions above. Forexample, the exact location of the t-PALP kringle and protease domainsin SEQ ID NO:2 may vary slightly (e.g., the address may “shift” by about1 to about 20 residues, more likely about 1 to about 5 residues)depending on the criteria used to define the domain.

Leader and Mature Sequences

The amino acid sequence of the complete t-PALP protein includes a leadersequence and a mature protein, as shown in SEQ ID NO:2. More inparticular, the present invention provides nucleic acid moleculesencoding a mature form of the t-PALP protein. Thus, according to thesignal hypothesis, once export of the growing protein chain across therough endoplasmic reticulum has been initiated, proteins secreted bymammalian cells have a signal or secretory leader sequence which iscleaved from the complete polypeptide to produce a secreted “mature”form of the protein. Most mammalian cells and even insect cells cleavesecreted proteins with the same specificity. However, in some cases,cleavage of a secreted protein is not entirely uniform, which results intwo or more mature species of the protein. Further, it has long beenknown that the cleavage specificity of a secreted protein is ultimatelydetermined by the primary structure of the complete protein, that is, itis inherent in the amino acid sequence of the polypeptide. Therefore,the present invention provides a nucleotide sequence encoding the maturet-PALP polypeptide having the amino acid sequence encoded by the cDNAclone contained in the host identified as ATCC Deposit No. 209023. Bythe “mature t-PALP polypeptide having the amino acid sequence encoded bythe cDNA clone in ATCC Deposit No. 209023” is meant the mature form(s)of the t-PALP protein produced by expression in a mammalian cell (e.g.,COS cells, as described below) of the complete open reading frameencoded by the human DNA sequence of the clone contained in the vectorin the deposited host.

In addition, methods for predicting whether a protein has a secretoryleader as well as the cleavage point for that leader sequence areavailable. For instance, the method of McGeoch (Virus Res. 3:271–286(1985)) uses the information from a short N-terminal charged region anda subsequent uncharged region of the complete (uncleaved) protein. Themethod of von Heinje (Nucleic Acids Res. 14:4683–4690 (1986)) uses theinformation from the residues surrounding the cleavage site, typicallyresidues −13 to +2 where +1 indicates the amino terminus of the matureprotein. The accuracy of predicting the cleavage points of knownmammalian secretory proteins for each of these methods is in the rangeof 75–80% (von Heinje, supra). However, the two methods do not alwaysproduce the same predicted cleavage point(s) for a given protein.

In the present case, the deduced amino acid sequence of the completet-PALP polypeptide was analyzed by a computer program PSORT, availablefrom Dr. Kenta Nakai of the Institute for Chemical Research, KyotoUniversity (see K. Nakai and M. Kanehisa, Genomics 14:897–911 (1992)),which is an expert system for predicting the cellular location of aprotein based on the amino acid sequence. As part of this computationalprediction of localization, the methods of McGeoch and von Heinje areincorporated. Thus, the computation analysis described above predicted asingle cleavable N-terminal signal sequence within the complete aminoacid sequence shown in SEQ ID NO:2.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. However, a nucleic acid contained in a clone that is a memberof a library (e.g., a genomic or cDNA library) that has not beenisolated from other members of the library (e.g., in the form of ahomogeneous solution containing the clone and other members of thelibrary) or a chromosome isolated or removed from a cell or a celllysate (e.g., a “chromosome spread”, as in a karyotype), is not“isolated” for the purposes of this invention. As discussed furtherherein, isolated nucleic acid molecules according to the presentinvention may be produced naturally, recombinantly, or synthetically.

In specific embodiments, the polynucleotides of the invention are lessthan 100000 kb, 50000 kb, 10000 kb, 1000 kb, 500 kb, 400 kb, 350 kb, 300kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb, 100 kb, 75 kb, 50 kb, 40 kb,30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 7.5 kb, or 5 kb in length. Inadditional specific embodiments, isolated polynucleotides of theinvention are less than 100000 kb, 50000 kb, 10000 kb, 1000 kb, 500 kb,400 kb, 350 kb, 300 kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb, 100 kb,75 kb, 50 kb, 40 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 7.5 kb, or 5 kbin length.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) with an initiationcodon at positions 124–126 of the nucleotide sequence shown in FIGS. 1A,1B, and 1C (SEQ ID NO:1).

Also included are DNA molecules comprising the coding sequence for thepredicted mature t-PALP protein shown at positions 1–242 of SEQUENCE IDNO:2.

In addition, isolated nucleic acid molecules of the invention includeDNA molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode the t-PALP protein. Of course, the genetic code andspecies-specific codon preferences are well known in the art. Thus, itwould be routine for one skilled in the art to generate the degeneratevariants described above, for instance, to optimize codon expression fora particular host (e.g., change codons in the human mRNA to thosepreferred by a bacterial host such as E. coli).

In another embodiment, the invention provides isolated nucleic acidmolecules encoding the t-PALP polypeptide having an amino acid sequenceencoded by the cDNA clone contained in the plasmid deposited as ATCCDeposit No. 209023 on May 8, 1997.

Preferably, this nucleic acid molecule will encode the maturepolypeptide encoded by the above-described deposited cDNA clone.

The invention further provides an isolated nucleic acid molecule havingthe nucleotide sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) orthe nucleotide sequence of the t-PALP cDNA contained in theabove-described deposited clone, or a nucleic acid molecule having asequence complementary to one of the above sequences. Such isolatedmolecules, particularly DNA molecules, are useful as probes for genemapping, by in situ hybridization with chromosomes, and for detectingexpression of the t-PALP gene in human tissue, for instance, by Northernblot analysis.

The present invention is further directed to nucleic acid moleculesencoding portions of the nucleotide sequences described herein as wellas to fragments of the isolated nucleic acid molecules described herein.In particular, the invention provides a polynucleotide having anucleotide sequence representing the portion of SEQ ID NO:1 whichconsists of positions 1–915 of SEQ ID NO:1.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:1 whichhave been determined from the following related cDNA clones: HTAAM28R(SEQ ID NO:4), HFKBA12R (SEQ ID NO:5), HAPBL24R (SEQ ID NO:6), HLMFG34R(SEQ ID NO:7), HHPGT42R (SEQ ID NO:8), HSSAX27R (SEQ ID NO:9), andHSSES93R (SEQ ID NO:10).

Further, the invention includes a polynucleotide comprising any portionof at least about 30 nucleotides, preferably at least about 50nucleotides, of SEQ ID NO:1 from residue 1 to 110 and from 630 to 750.More preferably, the invention includes a polynucleotide comprisingnucleotide residues 1 to 2000, 1 to 1500, 1 to 1000, 1 to 500, 1 to 250,250 to 2000, 250 to 1500, 250 to 1000, 250 to 500, 500 to 2000, 500 to1500, 500 to 1000, 1000 to 2000, and 1000 to 1500.

Moreover, representative examples of polynucleotide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide number 1–60, 61–123,1–168, 61–168, 124–168, 169–213, 214–258, 259–303, 304–348, 349–393,394–438, 439–483, 484–528, 529–573, 574–618, 619–663, 664–708, 709–753,754–798, 799–843, 844–888, 889–933, 934–978, 979–1023, 1024–1068,1069–1113, 1114–1158, 1159–1203, 1204–1248, 1249–1293, 1294–1338,1339–1383, 1384–1428, 1429–1473, 1474–1518, 1519–1563, 1564–1608,1609–1653, 1654–1698, 1699–1743, 1744–1788, 1789–1833, 1834–1878,1879–1923, 1924–1968, 1969–2013, 2014–2058, 2059–2103, 2104–2148,2149–2193, 2194–2238, 2239–2283, 2284–2328, or 2284–2329 of SEQ ID NO:1,or the complementary strand thereto, or the cDNA contained in thedeposited clone. In this context “about” includes the particularlyrecited ranges, and ranges larger or smaller by several (6, 5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini. Preferably,these fragments encode a polypeptide which has biological activity. Morepreferably, these polynucleotides can be used as probes or primers asdiscussed herein. Polynucleotides which hybridize to these nucleic acidmolecules under stringent hybridization conditions or lower stringencyconditions are also encompassed by the invention, as are polypeptidesencoded by these polynucleotides.

Representative examples of polynucleotide fragments of the invention,also include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide 1-123, 1-186, 1-195,1-375, 124-186, 124-195, 124-375, 187-261, 196-261, 196-375, 196-912,259-369, 259-375, 262-306, 262-333, 262-366, 262-375, 307-375, 328-345,322-351, 376-912, 376-777, 424-750, 748-807, 778-912, and 808-912 of SEQID NO:1, or the complementary strand thereto, or the cDNA contained inthe deposited clone.

More generally, by a fragment of an isolated nucleic acid moleculehaving the nucleotide sequence of the deposited cDNA or the nucleotidesequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) is intendedfragments at least about 15 nt, and more preferably at least about 20nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50–300 nt in length are also useful according to the presentinvention as are fragments corresponding to most, if not all, of thenucleotide sequence of the deposited cDNA or as shown in FIGS. 1A, 1B,and 1C (SEQ ID NO:1). By a fragment at least 20 nt in length, forexample, is intended fragments which include 20 or more contiguous basesfrom the nucleotide sequence of the deposited cDNA or the nucleotidesequence as shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1). Preferrednucleic acid fragments of the present invention include nucleic acidmolecules encoding epitope-bearing portions of the t-PALP polypeptide asidentified in FIG. 3 and described in more detail below.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, the cDNAclone contained in ATCC Deposit No. 209023. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30–70 (e.g., 35, 40, 45, 50, 55, 60, 65) nt of the referencepolynucleotide. These are useful as diagnostic probes and primers asdiscussed above and in more detail below.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in FIGS. 1A, 1B, and 1C (SEQ IDNO:1)). Of course, a polynucleotide which hybridizes only to a poly Asequence (such as the 3′ terminal poly(A) tract of the t-PALP cDNA shownin FIGS. 1A, 1B, and 1C (SEQ ID NO:1)), or to a complementary stretch ofT (or U) residues, would not be included in a polynucleotide of theinvention used to hybridize to a portion of a nucleic acid of theinvention, since such a polynucleotide would hybridize to any nucleicacid molecule containing a poly (A) stretch or the complement thereof(e.g., practically any double-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention whichencode a t-PALP polypeptide may include, but are not limited to thoseencoding the amino acid sequence of the predicted kringle domain, byitself, the amino acid sequence of the predicted protease domain, byitself, the amino acid sequence of the mature polypeptide, by itself;and the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 21 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro-protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences.

Also encoded by nucleic acids of the invention are the above proteinsequences together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; anadditional coding sequence which codes for additional amino acids, suchas those which provide additional functionalities.

Thus, the sequence encoding the polypeptide may be fused to a markersequence, such as a sequence encoding a peptide which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821–824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37: 767 (1984).As discussed below, other such fusion proteins include the t-PALP fusedto Fc at the N- or C-terminus.

In a preferred embodiment, the expression vectors pCMVFLAG5a orpFLAG-CMV-1 (available from Sigma, St. Louis, Mo., USA) are used for theexpression of t-PALP-FLAG fusion proteins of the invention. See,Andersson, S., et al., J. Biol. Chem. 264:8222–29 (1989); Thomsen, D.R., et al., Proc. Natl. Acad. Sci. USA, 81:659–63 (1984); and Kozak, M.,Nature 308:241 (1984) (each of which is hereby incorporated byreference). In this embodiment, t-PALP polynucleotides of the inventionare fused to a polynucleotide encoding a “FLAG” polypeptide. Thus, at-PALP-FLAG fusion protein is encompassed by the present invention. TheFLAG antigenic polypeptide may be fused to a t-PALP polypeptide of theinvention at either or both the amino- and carboxy-termini of the t-PALPprotein. In further preferred embodiments, a t-PALP-FLAG fusion proteinis detectable by anti-FLAG monoclonal antibodies (also available fromSigma).

In a further preferred embodiment, the FLAG polypeptide sequence isfused to the amino-terminus of amino acid residues Ser-22 throughAla-263 of the t-PALP amino acid sequence shown in FIGS. 1A, 1B, and 1C(which is identical to residues Ser-1 through Ala-242 of SEQ ID NO:2).This fusion protein is expressed from the pFLAG-CMV-1 expression vectorand is designated pFLAGCMV-1:t-PALP.S22-A263. This FLAG-t-PALPexpression construct will allow purification of t-PALP protein from thesupernatants of transiently transfected cell cultures by virtue of theamino-terminal FLAG tag. Moreover, any carboxy-terminal processing oft-PALP may also be detected and analyzed using this expressionconstruct.

In an additional preferred embodiment, the FLAG polypeptide sequence isfused to the carboxy-terminus of amino acid residues Met-1 throughAsp-165 of the t-PALP amino acid sequence shown in FIGS. 1A, 1B, and 1C(which is identical to residues Met-(−21) through Asp-144 of SEQ IDNO:2). This fusion protein is expressed from the pFLAG-CMV-5a expressionvector and is designated pFLAGCMV-5a:t-PALP.M1-D165. This FLAG-t-PALPexpression construct will allow purification of t-PALP protein from thesupernatants of transiently transfected cell cultures by virtue of theamino-terminal FLAG tag. Moreover, use of this expression construct willallow a determination of the predicted processed form of t-PALP byisolation via the FLAG tag and subsequent C-terminal peptide sequencing.Also, use of this expression construct will enable an analysis ofbiological and/or structural activities of a predicted processed form oft-PALP (i.e., amino acid residues 1–144 of SEQ ID NO:2).

Variant and Mutant Polynucleotides

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the t-PALP protein. Variants may occur naturally, such asa natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the t-PALP protein or portions thereof.Also especially preferred in this regard are conservative substitutions.For example, conservative amino acid substitutions of t-PALP can be madeby site directed changes which replace a particular amino acid with aconservative amino acid. Preferred conservative substitutions include:M1 replaced with A, G, I, L, S, T, or V; L2 replaced with A, G, I, S, T,M, or V; L3 replaced with A, G, I, S, T, M, or V; A4 replaced with G, I,L, S, T, M, or V; W5 replaced with F, or Y; V6 replaced with A, G, I, L,S, T, or M; Q7 replaced with N; A8 replaced with G, I, L, S, T, M, or V;F9 replaced with W, or Y; L10 replaced with A, G, I, S, T, M, or V; V11replaced with A, G, I, L, S, T, or M; S12 replaced with A, G, I, L, T,M, or V; N13 replaced with Q; M14 replaced with A, G, I, L, S, T, or V;L15 replaced with A, G, I, S, T, M, or V; L16 replaced with A, G, I, S,T, M, or V; A17 replaced with G, I, L, S, T, M, or V; E18 replaced withD; A19 replaced with G, I, L, S, T, M, or V; Y20 replaced with F, or W;G21 replaced with A, I, L, S, T, M, or V; S22 replaced with A, G, I, L,T, M, or V; G23 replaced with A, I, L, S, T, M, or V; G24 replaced withA, I, L, S, T, M, or V; F26 replaced with W, or Y; W27 replaced with F,or Y; D28 replaced with E; N29 replaced with Q; G30 replaced with A, I,L, S, T, M, or V; H31 replaced with K, or R; L32 replaced with A, G, I,S, T, M, or V; Y33 replaced with F, or W; R34 replaced with H, or K; E35replaced with D; D36 replaced with E; Q37 replaced with N; T38 replacedwith A, G, I, L, S, M, or V; S39 replaced with A, G, I, L, T, M, or V;A41 replaced with G, I, L, S, T, M, or V; G43 replaced with A, I, L, S,T, M, or V; L44 replaced with A, G, I, S, T, M, or V; R45 replaced withH, or K; L47 replaced with A, G, I, S, T, M, or V; N48 replaced with Q;W49 replaced with F, or Y; L50 replaced with A, G, I, S, T, M, or V; D51replaced with E; A52 replaced with G, I, L, S, T, M, or V; Q53 replacedwith N; S54 replaced with A, G, I, L, T, M, or V; G55 replaced with A,I, L, S, T, M, or V; L56 replaced with A, G, I, S, T, M, or V; A57replaced with G, I, L, S, T, M, or V; S58 replaced with A, G, I, L, T,M, or V; A59 replaced with G, I, L, S, T, M, or V; V61 replaced with A,G, I, L, S, T, or M; S62 replaced with A, G, I, L, T, M, or V; G63replaced with A, I, L, S, T, M, or V; A64 replaced with G, I, L, S, T,M, or V; G65 replaced with A, I, L, S, T, M, or V; N66 replaced with Q;H67 replaced with K, or R; S68 replaced with A, G, I, L, T, M, or V; Y69replaced with F, or W; R71 replaced with H, or K; N72 replaced with Q;D74 replaced with E; E75 replaced with D; D76 replaced with E; R78replaced with H, or K; G79 replaced with A, I, L, S, T, M, or V; W81replaced with F, or Y; Y83 replaced with F, or W; V84 replaced with A,G, I, L, S, T, or M; S85 replaced with A, G, I, L, T, M, or V; G86replaced with A, I, L, S, T, M, or V; E87 replaced with D; A88 replacedwith G, I, L, S, T, M, or V; G89 replaced with A, I, L, S, T, M, or V;V90 replaced with A, G, I, L, S, T, or M; E92 replaced with D; K93replaced with H, or R; R94 replaced with H, or K; E97 replaced with D;D98 replaced with E; L99 replaced with A, G, I, S, T, M, or V; R100replaced with H, or K; E103 replaced with D; T104 replaced with A, G, I,L, S, M, or V; T105 replaced with A, G, I, L, S, M, or V; S106 replacedwith A, G, I, L, T, M, or V; Q107 replaced with N; A108 replaced with G,I, L, S, T, M, or V; L109 replaced with A, G, I, S, T, M, or V; A111replaced with G, I, L, S, T, M, or V; F112 replaced with W, or Y; T113replaced with A, G, I, L, S, M, or V; T114 replaced with A, G, I, L, S,M, or V; E115 replaced with D; I116 replaced with A, G, L, S, T, M, orV; Q117 replaced with N; E118 replaced with D; A119 replaced with G, I,L, S, T, M, or V; S120 replaced with A, G, I, L, T, M, or V; E121replaced with D; G122 replaced with A, I, L, S, T, M, or V; G124replaced with A, I, L, S, T, M, or V; A125 replaced with G, I, L, S, T,M, or V; D126 replaced with E; E127 replaced with D; V128 replaced withA, G, I, L, S, T, or M; Q129 replaced with N; V130 replaced with A, G,I, L, S, T, or M; F131 replaced with W, or Y; A132 replaced with G, I,L, S, T, M, or V; A134 replaced with G, I, L, S, T, M, or V; N135replaced with Q; A136 replaced with G, I, L, S, T, M, or V; L137replaced with A, G, I, S, T, M, or V; A139 replaced with G, I, L, S, T,M, or V; R140 replaced with H, or K; S141 replaced with A, G, I, L, T,M, or V; E142 replaced with D; A143 replaced with G, I, L, S, T, M, orV; A144 replaced with G, I, L, S, T, M, or V; A145 replaced with G, I,L, S, T, M, or V; V146 replaced with A, G, I, L, S, T, or M; Q147replaced with N; V149 replaced with A, G, I, L, S, T, or M; I150replaced with A, G, L, S, T, M, or V; G151 replaced with A, I, L, S, T,M, or V; I152 replaced with A, G, L, S, T, M, or V; S153 replaced withA, G, I, L, T, M, or V; Q154 replaced with N; R155 replaced with H, orK; V156 replaced with A, G, I, L, S, T, or M; R157 replaced with H, orK; M158 replaced with A, G, I, L, S, T, or V; N159 replaced with Q; S160replaced with A, G, I, L, T, M, or V; K161 replaced with H, or R; E162replaced with D; K163 replaced with H, or R; K164 replaced with H, or R;D165 replaced with E; L166 replaced with A, G, I, S, T, M, or V; G167replaced with A, I, L, S, T, M, or V; T168 replaced with A, G, I, L, S,M, or V; L169 replaced with A, G, I, S, T, M, or V; G170 replaced withA, I, L, S, T, M, or V; Y171 replaced with F, or W; V172 replaced withA, G, I, L, S, T, or M; L173 replaced with A, G, I, S, T, M, or V; G174replaced with A, I, L, S, T, M, or V; I175 replaced with A, G, L, S, T,M, or V; T176 replaced with A, G, I, L, S, M, or V; M177 replaced withA, G, I, L, S, T, or V; M178 replaced with A, G, I, L, S, T, or V; V179replaced with A, G, I, L, S, T, or M; I180 replaced with A, G, L, S, T,M, or V; I181 replaced with A, G, L, S, T, M, or V; I182 replaced withA, G, L, S, T, M, or V; A183 replaced with G, I, L, S, T, M, or V; I184replaced with A, G, L, S, T, M, or V; G185 replaced with A, I, L, S, T,M, or V; A186 replaced with G, I, L, S, T, M, or V; G187 replaced withA, I, L, S, T, M, or V; I188 replaced with A, G, L, S, T, M, or V; I189replaced with A, G, L, S, T, M, or V; L190 replaced with A, G, I, S, T,M, or V; G191 replaced with A, I, L, S, T, M, or V; Y192 replaced withF, or W; S193 replaced with A, G, I, L, T, M, or V; Y194 replaced withF, or W; K195 replaced with H, or R; R196 replaced with H, or K; G197replaced with A, I, L, S, T, M, or V; K198 replaced with H, or R; D199replaced with E; L200 replaced with A, G, I, S, T, M, or V; K201replaced with H, or R; E202 replaced with D; Q203 replaced with N; H204replaced with K, or R; D205 replaced with E; Q206 replaced with N; K207replaced with H, or R; V208 replaced with A, G, I, L, S, T, or M; E210replaced with D; R211 replaced with H, or K; E212 replaced with D; M213replaced with A, G, I, L, S, T, or V; Q214 replaced with N; R215replaced with H, or K; I216 replaced with A, G, L, S, T, M, or V; T217replaced with A, G, I, L, S, M, or V; L218 replaced with A, G, I, S, T,M, or V; L220 replaced with A, G, I, S, T, M, or V; S221 replaced withA, G, I, L, T, M, or V; A222 replaced with G, I, L, S, T, M, or V; F223replaced with W, or Y; T224 replaced with A, G, I, L, S, M, or V; N225replaced with Q; T227 replaced with A, G, I, L, S, M, or V; E229replaced with D; I230 replaced with A, G, L, S, T, M, or V; V231replaced with A, G, I, L, S, T, or M; D232 replaced with E; E233replaced with D; K234 replaced with H, or R; T235 replaced with A, G, I,L, S, M, or V; V236 replaced with A, G, I, L, S, T, or M; V237 replacedwith A, G, I, L, S, T, or M; V238 replaced with A, G, I, L, S, T, or M;H239 replaced with K, or R; T240 replaced with A, G, I, L, S, M, or V;S241 replaced with A, G, I, L, T, M, or V; Q242 replaced with N; T243replaced with A, G, I, L, S, M, or V; V245 replaced with A, G, I, L, S,T, or M; D246 replaced with E; Q248 replaced with N; E249 replaced withD; G250 replaced with A, I, L, S, T, M, or V; S251 replaced with A, G,I, L, T, M, or V; T252 replaced with A, G, I, L, S, M, or V; L254replaced with A, G, I, S, T, M, or V; M255 replaced with A, G, I, L, S,T, or V; G256 replaced with A, I, L, S, T, M, or V; Q257 replaced withN; A258 replaced with G, I, L, S, T, M, or V; G259 replaced with A, I,L, S, T, M, or V; T260 replaced with A, G, I, L, S, M, or V; G262replaced with A, I, L, S, T, M, or V; or A263 replaced with G, I, L, S,T, M, or V.

The resulting constructs can be routinely screened for activities orfunctions described throughout the specification and known in the art.Preferably, the resulting constructs have an increased t-PALP activityor function (including dominant negative activities or functions), whilethe remaining t-PALP activities or functions are maintained. Morepreferably, the resulting constructs have more than one increased t-PALPactivity or function (including dominant negative activities orfunctions), while the remaining t-PALP activities or functions aremaintained.

Besides conservative amino acid substitution, variants of t-PALP include(i) substitutions with one or more of the non-conserved amino acidresidues, where the substituted amino acid residues may or may not beone encoded by the genetic code, or (ii) substitution with one or moreof amino acid residues having a substituent group, or (iii) fusion ofthe mature polypeptide with another compound, such as a compound toincrease the stability and/or solubility of the polypeptide (forexample, polyethylene glycol), or (iv) fusion of the polypeptide withadditional amino acids, such as, for example, an IgG Fc fusion regionpeptide, or leader or secretory sequence, or a sequence facilitatingpurification. Such variant polypeptides are deemed to be within thescope of those skilled in the art from the teachings herein.

For example, t-PALP polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331–340 (1967);Robbins et al., Diabetes 36: 838–845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307–377 (1993).)

For example, preferred non-conservative substitutions of t-PALP include:M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L2 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L3 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; A4 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; W5 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; V6 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q7replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;A8 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F9 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L10 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V11 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; S12 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; N13 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; M14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L15replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L16 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A17 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; E18 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; A19 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; Y20 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,V, P, or C; G21 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S22replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G23 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; G24 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; C25 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or P; F26 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; W27 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; D28 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; N29 replaced with D, E, H, K, R, A, G,I, L, S, T, M, V, F, W, Y, P, or C; G30 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; H31 replaced with D, E, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; L32 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; Y33 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; R34 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; E35 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; D36 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; Q37 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; T38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S39 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P40 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A41replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P42 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G43 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L44 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; R45 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; C46 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or P; L47 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; N48 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; W49 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; L50 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; D51 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; A52 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q53replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;S54 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G55 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L56 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A57 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; S58 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;A59 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P60 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V61replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S62 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; G63 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; A64 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; G65 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N66replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;H67 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;S68 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y69 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; C70 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; R71replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N72replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;P73 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; D74 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; E75 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; D76 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; P77 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or C; R78 replaced with D, E, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; G79 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; P80 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or C; W81 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,V, P, or C; C82 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, or P; Y83 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; V84 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; S85 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G86 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; E87 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A88 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; G89 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; V90 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; P91 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or C; E92 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; K93 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; R94 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; P95 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, or C; C96 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, or P; E97 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; D98 replaced with H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; L99 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; R100 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; C101 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or P; P102 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, or C; E103 replaced with H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; T104 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; T105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S106 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q107 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A108replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L109 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; P110 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A111 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; F112 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; T113 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; T114 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;E115 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; I116 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q117replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;E118 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; A119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S120replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E121 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G122 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P123 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G124 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; A125 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; D126 replaced with H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; E127 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; V128 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; Q129 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; V130 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;F131 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;A132 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P133 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A134replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N135 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A136 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L137 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P138 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; A139 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; R140 replaced with D, E, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; S141 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; E142 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; A143 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A144replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A145 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V146 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; Q147 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; P148 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, or C; V149 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; I150 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; G151 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I152replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S153 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; Q154 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; R155 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V156 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; R157 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; M158 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; N159 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; S160 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;K161 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;E162 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; K163 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; K164 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; D165 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; L166 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G167replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T168 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L169 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G170 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; Y171 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; V172 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L173replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G174 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; I175 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; T176 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; M177 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M178replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V179 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; I180 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; I181 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; I182 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A183replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I184 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; G185 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; A186 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; G187 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I188replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I189 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L190 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G191 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; Y192 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; S193 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y194replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K195replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R196replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G197replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K198 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D199 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L200 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; K201 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E202 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q203 replaced with D, E,H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; H204 replaced with D,E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D205 replaced with H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q206 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K207 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V208 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; C209 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; E210 replaced with H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R211 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E212 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M213 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; Q214 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R215 replaced with D, E,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I216 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; T217 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; L218 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; P219 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or C; L220 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S221replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A222 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; F223 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; T224 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; N225 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; P226 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, or C; T227 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; C228 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, or P; E229 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; I230 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; V231 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;D232 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; E233 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; K234 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; T235 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V236replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V237 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V238 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; H239 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; T240 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; S241 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q242replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;T243 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P244 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V245replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D246 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P247 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; Q248replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;E249 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; G250 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S251replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T252 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; P253 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L254 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; M255 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; G256 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;Q257 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; A258 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G259replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T260 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; P261 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G262 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; or A263 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C.

The resulting constructs can be routinely screened for activities orfunctions described throughout the specification and known in the art.Preferably, the resulting constructs have an increased t-PALP activityor function (including dominant negative activities or functions), whilethe remaining t-PALP activities or functions are maintained. Morepreferably, the resulting constructs have more than one increased t-PALPactivity or function (including dominant negative activities orfunctions), while the remaining t-PALP activities or functions aremaintained.

Additionally, more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9 and10) can be replaced with the substituted amino acids as described above(either conservative or nonconservative). The substituted amino acidscan occur in the full length, mature, and/or proprotein form of t-PALPprotein, as well as the N- and C-terminal deletion mutants, having thegeneral formulae m-n, m¹-n¹, m²-n², m³-n³ or m⁴-n⁴, listed below.

Most highly preferred are nucleic acid molecules encoding the matureprotein having the amino acid sequence shown in SEQ ID NO:2 or themature t-PALP amino acid sequence encoded by the deposited cDNA clone.

Most highly preferred are nucleic acid molecules encoding the proteasedomain of the protein having the amino acid sequence shown in SEQ IDNO:2 or the protease domain of the t-PALP amino acid sequence encoded bythe deposited cDNA clone.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding a full-length t-PALP polypeptide having the complete amino acidsequence in SEQ ID NO:2 excepting the N-terminal methionine (i.e.,positions −20 to 242 of SEQ ID NO:2) or the complete amino acid sequenceexcepting the N-terminal methionine encoded by the cDNA clone containedin the ATCC Deposit No. 209023; (b) a nucleotide sequence encoding thepredicted mature form of the t-PALP polypeptide having the amino acidsequence at positions 1 to 242 in SEQ ID NO:2 or as encoded by the cDNAclone contained in the ATCC Deposit No. 209023; (c) a nucleotidesequence encoding the predicted kringle domain of the t-PALP polypeptidehaving the amino acid sequence at positions 4 to 63 in SEQ ID NO:2 or asencoded by the cDNA clone contained in the ATCC Deposit No. 209023; (d)a nucleotide sequence encoding a polypeptide comprising the predictedprotease domain of the t-PALP polypeptide having the amino acid sequenceat positions 64 to 242 in SEQ ID NO:2 or as encoded by the cDNA clonecontained in the ATCC Deposit No. 209023; and (e) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d)or (e) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98%,99%, or 100% identical, to any of the nucleotide sequences in (a), (b),(c), (d) or (e) above, or a polynucleotide which hybridizes understringent hybridization conditions to a polynucleotide in (a), (b), (c),(d) or (e) above. In other words, these embodiments of the inventioninclude isolated nucleic acid molecules that comprise a polynucleotidehaving a nucleotide sequence which contains at most 10% differences, andmore preferably, at most 5%, 4%, 3%, 2%, 1%, or 0% differences, with anyof the nucleotide sequences in (a), (b), (c), (d) or (e) above, or apolynucleotide which hybridizes under stringent hybridization conditionsto a polynucleotide in (a), (b), (c), (d) or (e) above. Thispolynucleotide which hybridizes does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues. Anadditional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of a t-PALPpolypeptide having an amino acid sequence in (a), (b), (c) or (d) above.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production oft-PALP polypeptides or peptides by recombinant techniques.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to (or stated in another way,at most 10%, 5%, 4%, 3%, 2% or 1% different from) the nucleic acidsequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO: 1) or to the nucleicacid sequence of the deposited cDNA, irrespective of whether they encodea polypeptide having t-PALP activity. This is because even where aparticular nucleic acid molecule does not encode a polypeptide havingt-PALP activity, one of skill in the art would still know how to use thenucleic acid molecule, for instance, as a hybridization probe or apolymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving t-PALP activity include, inter alia, (1) isolating the t-PALPgene or allelic variants thereof in a cDNA library; (2) in situhybridization (e.g., “FISH”) to metaphase chromosomal spreads to provideprecise chromosomal location of the t-PALP gene, as described in Vermaet al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York (1988); and Northern Blot analysis for detecting t-PALP mRNAexpression in specific tissues.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a t-PALPpolypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five mismatches per each 100nucleotides of the reference nucleotide sequence encoding the t-PALPpolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. The reference (query) sequence may be the entirenucleotide sequence encoding t-PALP, as shown in FIGS. 1A and 1B (SEQ IDNO:1), or any t-PALP polynucleotide fragment as described herein.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequences shown in FIGS. 1A and 1B or to the nucleotidessequence of the deposited cDNA clone, or fragments thereof, can bedetermined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). Bestfit uses the local homology algorithmof Smith and Waterman to find the best segment of homology between twosequences (Advances in Applied Mathematics 2:482–489 (1981)). When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference nucleotide sequence and that gaps in homology ofup to 5% of the total number of nucleotides in the reference sequenceare allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag and colleagues(Comp. App. Biosci. 6:237–245 (1990)). In a sequence alignment the queryand subject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. The result of said global sequencealignment is in percent identity. Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions, a manual correction ismade to the results to take into consideration the fact that the FASTDBprogram does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example, a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatched/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%. In another example, a 90 base subjectsequence is compared with a 100 base query sequence. This time thedeletions are internal deletions so that there are no bases on the 5′ or3′ of the subject sequence which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only bases 5′ and 3′ of the subject sequencewhich are not matched/aligned with the query sequence are manuallycorrected for. No other manual corrections are made for the purposes ofthis embodiment.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to (or 10%, 5%, 4%, 3%, 2% or1% different from) the nucleic acid sequence shown in FIGS. 1A, 1B, and1C (SEQ ID NO:1) or to the nucleic acid sequence of the deposited cDNAwhich do, in fact, encode a polypeptide having t-PALP protein activity.By “a polypeptide having t-PALP activity” is intended polypeptidesexhibiting activity similar, but not necessarily identical, to anactivity of the mature t-PALP protein of the invention, as measured in aparticular biological assay. For example, the t-PALP protein of thepresent invention binds to fibrin. Such binding is assumed to mediatethe stimulation of plasminogen activation and the ultimate lysis of aplasma clot. The ability of t-PALP, or other related proteins, to bindto fibrin may be assayed in an in vitro analysis, as described by Kalyanand colleagues (J. Biol. Chem. 263:3971–3978; 1988). Briefly, a fibrinclot is generated by clotting fibrinogen by the addition of thrombin to1 unit/mL, incubating for 1 h at room temperature, and compacting bycentrifugation. The clot is then washed once with 50 mM Tris-HCl (pH7.4), 38 mM NaCl. Approximately 1000–2000 ng/mL of isolated t-PALP, oranother related protein, are then incubated with the above-describedplasminogen-free fibrin clot in a binding buffer consisting of 50 mMTris-HCl (pH 7.4), 38 mM NaCl, 100 micrograms/mL albumin, 1600micrograms/mL (˜5 micromolar) fibrinogen (plasminogen-free) for 1 h atroom temperature. Again, the clot is compacted by centrifugation andwashed once with 50 mM Tris-HCl (pH 7.4), 38 mM NaCl. The binding oft-PALP, or other related protein, to fibrin is then quantitated by gelelcetrophoresis and fibrin autography. Such fibrin-binding activity is auseful means of quantifying the ability of t-PALP, or a related protein,to bind to fibrin.

In addition, a general amidolytic activity of t-PALP, or another relatedprotein, may also be assessed through the use of a simple biochemicalassay also described by Kalyan and colleagues (J. Biol. Chem.263:3971–3978; 1988). Cleavage of a synthetic chromogenic substrate(S-2288) may be used to assess the general amidolytic activity oft-PALP, or another related protein. Hydrolysis of this compound producesp-nitroaniline which may be easily quantitated spectrophotometrically byits absorbance at 405 nm. Amidolytic reactions contain 150 mM Tris-HCl(ph 8.4), 100 micrograms/mL albumin, 0.01% Tween-80, 4 nM t-PALP, orother related protein, and 0.6 mM S-2288. Reactions are performed in inmicrotiter plates and the differential absorbance at 405–540 nm arerecorded at ten minute intervals up to 1 hour. Results are plotted asabsorbance versus time. This analysis can be enhanced with a slightalteration.

Since it is well-known that fibrin greatly enhances plasminogenactivation by t-PA and t-PALP, the generation of plasmin so formed canby conveniently measured by the slightly modified amidolytic assay. Inthis assay, the chromogenic substrate used is S-2251(D-Val-L-Ile-Lys-p-nitroanalide). Plasminogen activation reactionscontain 50 mM Tris-HCl (ph 7.4), 150 mM NaCl, 100 micrograms/mL albumin,0.01% Tween-80, 0.3 nM t-PALP, or other related protein, 0.6 mM S-2251,125 micrograms/mL soluble fibrin, and 1.5 micrograms/mL Glu-plasminogen.Reactions are performed in microtiter plates and are initiated by theaddition of plasminogen and S-2251. The differential absorbance at405–540 nm is recorded at 15 minute intervals and plotted as absorbanceversus time.

Further, the activity of t-PALP, or another related polypeptide, can beassessed by using a plasma clot lysis assay, essentially as describedKalyan and colleagues (J. Biol. Chem. 263:3971–3978; 1988). In thisanalysis, the ability of t-PALP, or another related polypeptide, to lyseradiolabeled preformed plasma clots are assessed by bathing clots inplasma containing an appropriate concentration of t-PALP, or anotherrelated polypeptide, and monitoring the release of degraded,radiolabeled fibrin. In this assay, 100 microliters of human citratedplasma is clotted in the presence of 0.5 microcuries ¹²⁵I-fibrinogen bythe addition of CaCl₂ to 25 micromolar and 2 units/mL thrombin. The clotis allowed to form at room temperature for 24 hours. Theradioactively-labeled clot is then bathed in 1 mL of plasma whichcontains a series of concentrations of t-PALP, or another relatedpolypeptide, (12.5 to 200 ng/mL); The reactions are shaken gently at 37°C. and samples are taken from the reactions at timepoints up to 24hours. Aliquots of each sample (10 microliters) are counted in a gcounter and expressed as the percent of total counts expected fromcomplete clot lysis.

t-PALP protein binds fibrin, has amidolytic activity, and can lyse aplasma clot in a dose-dependent manner in the above-described assays.Thus, “a polypeptide having t-PALP protein activity” includespolypeptides that also exhibit any of the same activities in theabove-described assays in a dose-dependent manner. Although the degreeof dose-dependent activity need not be identical to that of the t-PALPprotein, preferably, “a polypeptide having t-PALP protein activity” willexhibit substantially similar dose-dependence in a given activity ascompared to the t-PALP protein (i.e., the candidate polypeptide willexhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity relative to thereference t-PALP protein).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to (or 10%, 5%, 4%, 3%, 2% or 1% different from)the nucleic acid sequence of the deposited cDNA or the nucleic acidsequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) will encode apolypeptide “having t-PALP protein activity.” In fact, since degeneratevariants of these nucleotide sequences all encode the same polypeptide,this will be clear to the skilled artisan even without performing theabove described comparison assay. It will be further recognized in theart that, for such nucleic acid molecules that are not degeneratevariants, a reasonable number will also encode a polypeptide havingt-PALP protein activity. This is because the skilled artisan is fullyaware of amino acid substitutions that are either less likely or notlikely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid), as furtherdescribed below.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of t-PALPpolypeptides or fragments thereof by recombinant techniques. The vectormay be, for example, a phage, plasmid, viral or retroviral vector.Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe transcripts expressed by the constructs will preferably include atranslation initiating codon at the beginning and a termination codon(UAA, UGA or UAG) appropriately positioned at the end of the polypeptideto be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

Among vectors preferred for use in bacteria include pHE4-5 (ATCCAccession No. 209311) (and/or other related pHE-type vectors) pQE70,pQE60 and pQE-9, available from QIAGEN, Inc., supra; pBS vectors,Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,pRIT5 available from Pharmacia. Among preferred eukaryotic vectors arepWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; andpSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitablevectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to stabilize andpurify proteins. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobulin molecules together with another humanprotein or part thereof. In many cases, the Fc part in a fusion proteinis thoroughly advantageous for use in therapy and diagnosis and thusresults, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described. This is thecase when Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example when the fusion protein is to be used as antigenfor immunizations. In drug discovery, for example, human proteins, suchas hIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., J Molecular Recognition 8:52–58 (1995) and K.Johanson et al., J. Biol. Chem. 270:9459–9471 (1995).

The t-PALP protein can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Polypeptides of the presentinvention include: products purified from natural sources, includingbodily fluids, tissues and cells, whether directly isolated or cultured;products of chemical synthetic procedures; and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

Polypeptides and Fragments

The invention further provides an isolated t-PALP polypeptide having theamino acid sequence encoded by the deposited cDNA, or the amino acidsequence in SEQ ID NO:2, or a peptide or polypeptide comprising aportion of the above polypeptides.

Variant and Mutant Polypeptides

To improve or alter the characteristics of t-PALP polypeptides, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins or“muteins including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

N-Terminal and C-Terminal Deletion Mutants

For instance, for many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron and colleagues (J. Biol. Chem.,268:2984–2988; 1993) reported modified KGF proteins that had heparinbinding activity even if 3, 8, or 27 amino-terminal amino acid residueswere missing. In the present case, since the protein of the invention isrelated to t-PA, deletions of N-terminal amino acids up to the serine atposition 64 of SEQ ID NO:2 may retain some proteolytic activity.Polypeptides having further N-terminal deletions including the serineresidue in SEQ ID NO:2 would not be expected to retain such biologicalactivities because it is known that this residue in t-PA is in thebeginning of the conserved protease domain required for its observedproteolytic activity.

However, even if deletion of one or more amino acids from the N-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other functional activities may still beretained (for example, biological activities, ability to catalyzeproteolysis, ability to bind t-PALP receptors). Thus, the ability of theshortened protein to induce and/or bind to antibodies which recognizethe complete or mature of the protein generally will be retained whenless than the majority of the residues of the complete or mature proteinare removed from the N-terminus. Whether a particular polypeptidelacking N-terminal residues of a complete protein retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thata t-PALP mutein with a large number of deleted N-terminal amino acidresidues may retain some biological or immungenic activities. In fact,peptides composed of as few as six t-PALP amino acid residues may oftenevoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence of the t-PALP shown in SEQ ID NO:2, up to the serine residue atposition number 64, and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising, oralternatively consisting of, the amino acid sequence of residues n¹–242of SEQ ID NO:2, where n¹ is an integer in the range of (−)21 to 64, and64 is the position of the first residue from the N-terminus of thecomplete t-PALP polypeptide (shown in SEQ ID NO:2) believed to berequired for proteolytic activity of the t-PALP protein.

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues of 1 to 242, 2to 242, 3 to 242, 4 to 242, 5 to 242, 6 to 242, 7 to 242, 8 to 242, 9 to242, 10 to 242, 11 to 242, 12 to 242, 13 to 242, 14 to 242, 15 to 242,16 to 242, 17 to 242, 18 to 242, 19 to 242, 20 to 242, 21 to 242, 22 to242, 23 to 242, 24 to 242, 25 to 242, 26 to 242, 27 to 242, 28 to 242,29 to 242, 30 to 242, 31 to 242, 32 to 242, 33 to 242, 34 to 242, 35 to242, 36 to 242, 37 to 242, 38 to 242, 39 to 242, 40 to 242, 41 to 242,42 to 242, 43 to 242, 44 to 242, 45 to 242, 46 to 242, 47 to 242, 48 to242, 49 to 242, 50 to 242, 51 to 242, 52 to 242, 53 to 242, 54 to 242,55 to 242, 56 to 242, 57 to 242, 58 to 242, 59 to 242, 60 to 242, 61 to242, 62 to 242 or 63 to 242 of SEQ ID NO:2. Polynucleotides encodingthese polypeptides also are provided.

Moreover, a signal sequence may be added to these N-terminal deletioncontructs. For example, amino acids Met(−)21 to Ser(+)1 of SEQ ID NO:2,amino acids Leu(−)20 to Ser(+)1 of SEQ ID NO:2, amino acids Leu(−)19 toSer(+)1 of SEQ ID NO:2, amino acids Ala(−)18 to Ser(+)1 of SEQ ID NO:2,amino acids Trp(−)17 to Ser(+)1 of SEQ ID NO:2, amino acids Val(−)16 toSer(+)1 of SEQ ID NO:2, amino acids Gln(−)15 to Ser(+)1 of SEQ ID NO:2,amino acids Ala(−)14 to Ser(+)1 of SEQ ID NO:2, amino acids Phe(−)13 toSer(+)1 of SEQ ID NO:2, amino acids Leu(−)12 to Ser(+)1 of SEQ ID NO:2,amino acids Val(−)11 to Ser(+)1 of SEQ ID NO:2, amino acids Ser(−)10 toSer(+)1 of SEQ ID NO:2, amino acids Asn(−)9 to Ser(+)1 of SEQ ID NO:2,amino acids Met(−)8 to Ser(+)1 of SEQ ID NO:2, amino acids Leu(−)7 toSer(+)1 of SEQ ID NO:2, amino acids Leu(−)6 to Ser(+)1 of SEQ ID NO:2,amino acids Ala(−)5 to Ser(+)1 of SEQ ID NO:2, amino acids Glu(−)4 toSer(+)1 of SEQ ID NO:2, amino acids Ala(−)3 to Ser(+)1 of SEQ ID NO:2,amino acids Tyr(−)2 to Ser(+)1 of SEQ ID NO:2 or amino acids Gly(−)1 toSer(+)1 of SEQ ID NO:2 can be added or fused to the N-terminus of eachdeletion construct listed above.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, Interferon-gamma shows up to ten timeshigher activities by deleting 8–10 amino acid residues from the carboxyterminus of the protein (Dobeli et al., (1988) J Biotechnol. 7:199–216).In the present case, since the protein of the invention is a member ofthe serine protease or t-PA polypeptide families, deletions ofC-terminal amino acids up to the serine at position 230 of SEQ ID NO:2may retain some of the observed proteolytic activity of thecarboxy-terminal protease domain of t-PA.

However, even if deletion of one or more amino acids from the C-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other functional activities may still beretained (for example, biological activities, ability to catalyzeproteolysis, ability to bind t-PALP receptors). Thus, the ability of theshortened protein to induce and/or bind to antibodies which recognizethe complete or mature form of the protein generally will be retainedwhen less than the majority of the residues of the complete or matureprotein are removed from the C-terminus. Whether a particularpolypeptide lacking C-terminal residues of a complete protein retainssuch immunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thata t-PALP mutein with a large number of deleted C-terminal amino acidresidues may retain some biological or immungenic activities. In fact,peptides composed of as few as six t-PALP amino acid residues may oftenevoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the t-PALP shown in SEQ ID NO:2, up to the serine residue atposition 230 of SEQ ID NO:2, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptideshaving the amino acid sequence of residues −20–m¹ of the amino acidsequence in SEQ ID NO:2, where m¹ is any integer in the range of 230 to241, and residue serine is the position of the first residue from theC-terminus of the complete t-PALP polypeptide (shown in SEQ ID NO:2)believed to be required for protease activity of the t-PALP protein.

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues −20–230,−20–231, −20–232, −20–233, −20–234, −20–235, −20–236, −20–237, −20–238,−20–239, −20–240, −20–241, −20–242 of SEQ ID NO:2. Polynucleotidesencoding these polypeptides also are provided.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues n¹–m¹ of SEQ ID NO:2, where n¹and m¹ are integers as described above.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete t-PALP amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 209023, wherethis portion excludes any integer of amino acid residues from 1 to about82 amino acids from the amino terminus of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209023,or any integer of amino acid residues from 1 to about 13 amino acidsfrom the carboxy terminus, or any combination of the above aminoterminal and carboxy terminal deletions, of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209023.Polynucleotides encoding all of the above deletion mutant polypeptideforms also are provided.

The present application is also directed to proteins containingpolypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to thet-PALP polypeptide sequence set forth herein as m¹–n¹. In preferredembodiments, the application is directed to proteins containingpolypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical topolypeptides having the amino acid sequence of the specific t-PALP N-and C-terminal deletions recited herein. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete t-PALP amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 209023, wherethis portion excludes from 1 to about 63 amino acids from the aminoterminus of the complete amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 209023, or from 1 to about 11 amino acidsfrom the carboxy terminus, or any combination of the above aminoterminal and carboxy terminal deletions, of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209023.Polynucleotides encoding all of the above deletion mutant polypeptideforms also are provided.

As mentioned above, even if deletion of one or more amino acids from theN-terminus of a protein results in modification of loss of one or morebiological functions of the protein, other functional activities maystill be retained (for example, biological activities, ability tocatalyze proteolysis, ability to bind t-PALP receptor). Thus, theability of the shortened t-PALP mutein to induce and/or bind toantibodies which recognize the complete or mature of the proteingenerally will be retained when less than the majority of the residuesof the complete or mature protein are removed from the N-terminus.Whether a particular polypeptide lacking N-terminal residues of acomplete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that a t-PALP mutein with a large number ofdeleted N-terminal amino acid residues may retain some biological orimmungenic activities. In fact, peptides composed of as few as sixt-PALP amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence of the t-PALP shown in SEQ ID NO:2, up to the alanine residueat position number 258 (numbering as shown in FIGS. 1A, 1B, and 1C;A-258 is A-237 in SEQ ID NO:2), and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues n²–258 of FIGS. 1A, 1B,and 1C (n²–237 of SEQ ID NO:2), where n is an integer in the range of2–258 (−21–258 of SEQ ID NO:2), and 258 is the position of the firstresidue from the N-terminus of the complete t-PALP polypeptide (shown asresidue 237 in SEQ ID NO:2) believed to be required for at leastimmunogenic activity of the t-PALP protein.

More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues of L-2 to A-263; L-3 to A-263; A-4 to A-263; W-5 toA-263; V-6 to A-263; Q-7 to A-263; A-8 to A-263; F-9 to A-263; L-10 toA-263; V-11 to A-263; S-12 to A-263; N-13 to A-263; M-14 to A-263; L-15to A-263; L-16 to A-263; A-17 to A-263; E-18 to A-263; A-19 to A-263;Y-20 to A-263; G-21 to A-263; S-22 to A-263; G-23 to A-263; G-24 toA-263; C-25 to A-263; F-26 to A-263; W-27 to A-263; D-28 to A-263; N-29to A-263; G-30 to A-263; H-31 to A-263; L-32 to A-263; Y-33 to A-263;R-34 to A-263; E-35 to A-263; D-36 to A-263; Q-37 to A-263; T-38 toA-263; S-39 to A-263; P-40 to A-263; A-41 to A-263; P-42 to A-263; G-43to A-263; L-44 to A-263; R-45 to A-263; C-46 to A-263; L-47 to A-263;N-48 to A-263; W-49 to A-263; L-50 to A-263; D-51 to A-263; A-52 toA-263; Q-53 to A-263; S-54 to A-263; G-55 to A-263; L-56 to A-263; A-57to A-263; S-58 to A-263; A-59 to A-263; P-60 to A-263; V-61 to A-263;S-62 to A-263; G-63 to A-263; A-64 to A-263; G-65 to A-263; N-66 toA-263; H-67 to A-263; S-68 to A-263; Y-69 to A-263; C-70 to A-263; R-71to A-263; N-72 to A-263; P-73 to A-263; D-74 to A-263; E-75 to A-263;D-76 to A-263; P-77 to A-263; R-78 to A-263; G-79 to A-263; P-80 toA-263; W-81 to A-263; C-82 to A-263; Y-83 to A-263; V-84 to A-263; S-85to A-263; G-86 to A-263; E-87 to A-263; A-88 to A-263; G-89 to A-263;V-90 to A-263; P-91 to A-263; E-92 to A-263; K-93 to A-263; R-94 toA-263; P-95 to A-263; C-96 to A-263; E-97 to A-263; D-98 to A-263; L-99to A-263; R-100 to A-263; C-101 to A-263; P-102 to A-263; E-103 toA-263; T-104 to A-263; T-105 to A-263; S-106 to A-263; Q-107 to A-263;A-108 to A-263; L-109 to A-263; P-110 to A-263; A-111 to A-263; F112 toA-263; T-113 to A-263; T-114 to A-263; E-115 to A-263; I-116 A-263;G-122 to A-263; P-123 to A-263; G-124 to A-263; A-125 to A-263; D-126 toA-263; E-127 to A-263; V-128 to A-263; Q-129 to A-263; V-130 to A-263;F-131 to A-263; A-132 to A-263; P-133 to A-263; A-134 to A-263; N-135 toA-263; A-136 to A-263; L-137 to A-263; P-138 to A-263; A-139 to A-263;R-140 to A-263; S-141 to A-263; E-142 to A-263; A-143 to A-263; A-144 toA-263; A-145 to A-263; V-146 to A-263; Q-147 to A-263; P-148 to A-263;V-149 to A-263; I-150 to A-263; G-151 to A-263; I-152 to A-263; S-153 toA-263; Q-154 to A-263; R-155 to A-263; V-156 to A-263; R-157 to A-263;M-158 to A-263; N-159 to A-263; S-160 to A-263; K-161 to A-263; E-162 toA-263; K-163 to A-263; K-164 to A-263; D-165 to A-263; L-166 to A-263;G-167 to A-263; T-168 to A-263; L-169 to A-263; G-170 to A-263; Y-171 toA-263; V-172 to A-263; L-173 to A-263; G-174 to A-263; I-175 to A-263;T-176 to A-263; M-177 to A-263; M-178 to A-263; V-179 to A-263; I-180 toA-263; I-181 to A-263; I-182 to A-263; A-183 to A-263; I-184 to A-263;G-185 to A-263; A-186 to A-263; G-187 to A-263; I-188 to A-263; I-189 toA-263; L-190 to A-263; G-191 to A-263; Y-192 to A-263; S-193 to A-263;Y-194 to A-263; K-195 to A-263; R-196 to A-263; G-197 to A-263; K-198 toA-263; D-199 to A-263; L-200 to A-263; K-201 to A-263; E-202 to A-263;Q-203 to A-263; H-204 to A-263; D-205 to A-263; Q-206 to A-263; K-207 toA-263; V-208 to A-263; C-209 to A-263; E-210 to A-263; R-211 to A-263;E-212 to A-263; M-213 to A-263; Q-214 to A-263; R-215 to A-263; I-216 toA-263; T-217 to A-263; L-218 to A-263; P-219 to A-263; L-220 to A-263;S-221 to A-263; A-222 to A-263; F-223 to A-263; T-224 to A-263; N-225 toA-263; P-226 to A-263; T-227 to A-263; C-228 to A-263; E-229 to A-263;I-230 to A-263; V-231 to A-263; D-232 to A-263; E-233 to A-263; K-234 toA-263; T-235 to A-263; V-236 to A-263; V-237 to A-263; V-238 to A-263;H-239 to A-263; T-240 to A-263; S-241 to A-263; Q-242 to A-263; T-243 toA-263; P-244 to A-263; V-245 to A-263; D-246 to A-263; P-247 to A-263;Q-248 to A-263; E-249 to A-263; G-250 to A-263; S-251 to A-263; T-252 toA-263; P-253 to A-263; L-254 to A-263; M-255 to A-263; G-256 to A-263;Q-257 to A-263; and A-258 to A-263 of the t-PALP sequence shown in SEQID NO:2 using the numbering scheme of FIGS. 1A, 1B, and 1C.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activitiesmay still be retained (for example, biological activities, ability tocatalyze proteolysis, ability to bind t-PALP receptor). Thus, theability of the shortened t-PALP mutein to induce and/or bind toantibodies which recognize the complete or mature of the proteingenerally will be retained when less than the majority of the residuesof the complete or mature protein are removed from the C-terminus.Whether a particular polypeptide lacking C-terminal residues of acomplete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that a t-PALP mutein with a large number ofdeleted C-terminal amino acid residues may retain some biological orimmungenic activities. In fact, peptides composed of as few as sixt-PALP amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the t-PALP shown in SEQ ID NO:2, up to the valine residue atposition number 6 (numbering as shown in FIGS. 1A, 1B, and 1C; thevaline at position 6 is the valine at position −14 in SEQ ID NO:2), andpolynucleotides encoding such polypeptides. In particular, the presentinvention provides polypeptides comprising the amino acid sequence ofresidues 1–m² of FIGS. 1A, 1B, and 1C (−21–m² of SEQ ID NO:2), where m²is an integer in the range of 7–263 (−13–242 of SEQ ID NO:2), and 6 isthe position of the first residue from the C-terminus of the completet-PALP polypeptide (shown as residue −14 in SEQ ID NO:2) believed to berequired for at least immunogenic activity of the t-PALP protein.

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues M-1 to G-262;M-1 to P-261; M-1 to T-260; M-1 to G-259; M-1 to A-258; M-1 to Q-257;M-1 to G-256; M-1 to M-255; M-1 to L-254; M-1 to P-253; M-1 to T-252;M-1 to S-251; M-1 to G-250; M-1 to E-249; M-1 to Q-248; M-1 to P-247;M-1 to D-246; M-1 to V-245; M-1 to P-244; M-1 to T-243; M-1 to Q-242;M-1 to S-241; M-1 to T-240; M-1 to H-239; M-1 to V-238; M-1 to V-237;M-1 to V-236; M-1 to T-235; M-1 to K-234; M-1 to E-233; M-1 to D-232;M-1 to V_231; M-1 to I-230; M-1 to E-229; M-1 to C-228; M-1 to T-227;M-1 to P-226; M-1 to N-225; M-1 to T-224; M-1 to F-223; M-1 to A-222;M-1 to S-221; M-1 to L-220; M-1 to P-219; M-1 to L-218; M-1 to T-217;M-1 to I-216; M-1 to R-215; M-1 to Q-214; M-1 to M-213; M-1 to E-212;M-1 to R-211; M-1 to E-210; M-1 to C-209; M-1 to V-208; M-1 to K-207;M-1 to Q-206; M-1 to D-205; M-1 to H-204; M-1 to Q-203; M-1 to E-202;M-1 to K-201; M-1 to L-200; M-1 to D-199; M-1 to K-198; M-1 to G-197;M-1 to R-196; M-1 to K-195; M-1 to Y-194; M-1 to S-193; M-1 to Y-192;M-1 to G-191; M-1 to L-190; M-1 to I-189; M-1 to I-188; M-1 to G-187;M-1 to A-186; M-1 to G-185; M-1 to 1-184; M-1 to A-183; M-1 to 1-182;M-1 to 1-181; M-1 to 1-180; M-1 to V-179; M-1 to M-178; M-1 to M-177;M-1 to T-176; M-1 to 1-175; M-1 to G-174; M-1 to L-173; M-1 to V-172;M-1 to Y-171; M-1 to G-170; M-1 to L-169; M-1 to T-168; M-1 to G-167;M-1 to L-166; M-1 to D-165; M-1 to K-164; M-1 to K-163; M-1 to E-162;M-1 to K-161; M-1 to S-160; M-1 to N-159; M-1 to M-158; M-1 to R-157;M-1 to V-156; M-1 to R-155; M-1 to Q-154; M-1 to S-153; M-1 to I-152;M-1 to G-151; M-1 to I-150; M-1 to V-149; M-1 to P-148; M-1 to Q-147;M-1 to V-146; M-1 to A-145; M-1 to A-144; M-1 to A-143; M-1 to E-142;M-1 to S-141; M-1 to R-140; M-1 to A-139; M-1 to P-138; M-1 to L-137;M-1 to A-136; M-1 to N-135; M-1 to A-134; M-1 to P-133; M-1 to A-132;M-1 to F-131; M-1 to V-130; M-1 to Q-129; M-1 to V-128; M-1 to E-127;M-1 to D-126; M-1 to A-125; M-1 to G-124; M-1 to P-123; M-1 to G-122;M-1 to E-121; M-1 to S-120; M-1 to A-119; M-1 to E-118; M-1 to Q-117;M-1 to I-116; M-1 to E-115; M-1 to T-114; M-1 to T-113; M-1 to F-112;M-1 to A-111; M-1 to P-110; M-1 to L-109; M-1 to A-108; M-1 to Q-107;M-1 to S-106; M-1 to T-105; M-1 to T-104; M-1 to E-103; M-1 to P-102;M-1 to C-101; M-1 to R-100; M-1 to L-99; M-1 to D-98; M-1 to E-97; M-1to C-96; M-1 to P-95; M-1 to R-94; M-1 to K-93; M-1 to E-92; M-1 toP-91; M-1 to V-90; M-1 to G-89; M-1 to A-88; M-1 to E-87; M-1 to G-86;M-1 to S-85; M-1 to V-84; M-1 to Y-83; M-1 to C-82; M-1 to W-81; M-1 toP-80; M-1 to G-79; M-1 to R-78; M-1 to P-77; M-1 to D-76; M-1 to E-75;M-1 to D-74; M-1 to P-73; M-1 to N-72; M-1 to R-71; M-1 to C-70; M-1 toY-69; M-1 to S-68; M-1 to H-67; M-1 to N-66; M-1 to G-65; M-1 to A-64;M-1 to G-63; M-1 to S-62; M-1 to V-61; M-1 to P-60; M-1 to A-59; M-1 toS-58; M-1 to A-57; M-1 to L-56; M-1 to G-55; M-1 to S-54; M-1 to Q-53;M-1 to A-52; M-1 to D-51; M-1 to L-50; M-1 to W-49; M-1 to N-48; M-1 toL-47; M-1 to C-46; M-1 to R-45; M-1 to L-44; M-1 to G-43; M-1 to P-42;M-1 to A-41; M-1 to P-40; M-1 to S-39; M-1 to T-38; M-1 to Q-37; M-1 toD-36; M-1 to E-35; M-1 to R-34; M-1 to Y-33; M-1 to L-32; M-1 to H-31;M-1 to G-30; M-1 to N-29; M-1 to D-28; M-1 to W-27; M-1 to F-26; M-1 toC-25; M-1 to G-24; M-1 to G-23; M-1 to S-22; M-1 to G-21; M-1 to Y-20;M-1 to A-19; M-1 to E-18; M-1 to A-17; M-1 to L-16; M-1 to L-15; M-1 toM-14; M-1 to N-13; M-1 to S-12; M-1 to V-11; M-1 to L-10; M-1 to F-9;M-1 to A-8; M-1 to Q-7; and M-1 to V-6 of the t-PALP sequence shown inSEQ ID NO:2 using the numbering scheme of FIGS. 1A, 1B, and 1C.Polynucleotides encoding these polypeptides also are provided.

Moreover, a signal sequence may be added to these C-terminal deletioncontructs. For example, amino acids Met(−)21 to Ser(+)1 of SEQ ID NO:2,amino acids Leu(−)20 to Ser(+)1 of SEQ ID NO:2, amino acids Leu(−)19 toSer(+)1 of SEQ ID NO:2, amino acids Ala(−)18 to Ser(+)1 of SEQ ID NO:2,amino acids Trp(−)17 to Ser(+)1 of SEQ ID NO:2, amino acids Val(−)16 toSer(+)1 of SEQ ID NO:2, amino acids Gln(−)15 to Ser(+)1 of SEQ ID NO:2,amino acids Ala(−)14 to Ser(+)1 of SEQ ID NO:2, amino acids Phe(−)13 toSer(+)1 of SEQ ID NO:2, amino acids Leu(−)12 to Ser(+)1 of SEQ ID NO:2,amino acids Val(−)11 to Ser(+)1 of SEQ ID NO:2, amino acids Ser(−)10 toSer(+)1 of SEQ ID NO:2, amino acids Asn(−)9 to Ser(+)1 of SEQ ID NO:2,amino acids Met(−)8 to Ser(+)1 of SEQ ID NO:2, amino acids Leu(−)7 toSer(+)1 of SEQ ID NO:2, amino acids Leu(−)6 to Ser(+)1 of SEQ ID NO:2,amino acids Ala(−)5 to Ser(+)1 of SEQ ID NO:2, amino acids Glu(−)4 toSer(+)1 of SEQ ID NO:2, amino acids Ala(−)₃ to Ser(+)1 of SEQ ID NO:2,amino acids Tyr(−)2 to Ser(+)1 of SEQ ID NO:2, and amino acids Gly(−)1to Ser(+)1 of SEQ ID NO:2 can be added or fused to the N-terminus ofeach deletion construct listed above.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues n to m² of SEQ ID NO:2, where n²and m² are integers as described above.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete t-PALP amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 209023, wherethis portion excludes any integer of amino acid residues from 1 to about232 amino acids from the amino terminus of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209023,or any integer of amino acid residues from 1 to about 232 amino acidsfrom the carboxy terminus, or any combination of the above aminoterminal and carboxy terminal deletions, of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209023.Polynucleotides encoding all of the above deletion mutant polypeptideforms also are provided.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the kringledomain (Ser-1 to Val-63) of t-PALP shown in SEQ ID NO:2, up to theglycine residue at position number 58, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising, or alternatively consisting of, the amino acid sequence ofresidues n³ to 63 of SEQ ID NO:2, where n³ is an integer in the range of1 to 58 (SEQ ID NO:2).

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues 1 to 63; 2 to63; 3 to 63; 4 to 63; 5 to 63; 6 to 63; 7 to 63; 8 to 63; 9 to 63; 10 to63; 11 to 63; 12 to 63; 13 to 63; 14 to 63; 15 to 63; 16 to 63; 17 to63; 18 to 63; 19 to 63; 20 to 63; 21 to 63; 22 to 63; 23 to 63; 24 to63; 25 to 63; 26 to 63; 27 to 63; 28 to 63; 29 to 63; 30 to 63; 31 to63; 32 to 63; 33 to 63; 34 to 63; 35 to 63; 36 to 63; 37 to 63; 38 to63; 39 to 63; 40 to 63; 41 to 63; 42 to 63; 43to 63; 44 to 63; 45 to 63;46 to 63; 47 to 63; 48 to 63; 48 to 63; 49 to 63; 50 to 63; 51 to 63; 52to 63; 53 to 63; 54 to 63; 55 to 63; 56 to 63; 57 to 63; and 58 to 63 ofSEQ ID NO:2. Polynucleotides encoding these polypeptides also areprovided.

Moreover, a signal sequence may be added to these N-terminal deletioncontructs. For example, amino acids Met(−)21 to Ser(+)1 of SEQ ID NO:2,amino acids Leu(−)20 to Ser(+)1 of SEQ ID NO:2, amino acids Leu(−)19 toSer(+)1 of SEQ ID NO:2, amino acids Ala(−)18 to Ser(+)1 of SEQ ID NO:2,amino acids Trp(−)17 to Ser(+)1 of SEQ ID NO:2, amino acids Val(−)16 toSer(+)1 of SEQ ID NO:2, amino acids Gln(−)15 to Ser(+)1 of SEQ ID NO:2,amino acids Ala(−)14 to Ser(+)1 of SEQ ID NO:2, amino acids Phe(−)13 toSer(+)1 of SEQ ID NO:2, amino acids Leu(−)12 to Ser(+)1 of SEQ ID NO:2,amino acids Val(−)11 to Ser(+)1 of SEQ ID NO:2, amino acids Ser(−)10 toSer(+)1 of SEQ ID NO:2, amino acids Asn(−)9 to Ser(+)1 of SEQ ID NO:2,amino acids Met(−)8 to Ser(+)1 of SEQ ID NO:2, amino acids Leu(−)7 toSer(+)1 of SEQ ID NO:2, amino acids Leu(−)6 to Ser(+)1 of SEQ ID NO:2,amino acids Ala(−)5 to Ser(+)1 of SEQ ID NO:2, amino acids Glu(−)4 toSer(+)1 of SEQ ID NO:2, amino acids Ala(−)₃ to Ser(+)1 of SEQ ID NO:2,amino acids Tyr(−)2 to Ser(+)1 of SEQ ID NO:2, and amino acids Gly(−)1to Ser(+)1 of SEQ ID NO:2 can be added or fused to the N-terminus ofeach deletion construct listed above.

The present invention further provides polypeptides having one or moreamino acid residues deleted from the carboxy terminus of the kringledomain (Ser-1 to Val-63) of t-PALP (shown in SEQ ID NO:2), up to thetryptophan residue at position 6 of SEQ ID NO:2, and polynucleotidesencoding such polypeptides. In particular, the present inventionprovides polypeptides having the amino acid sequence of residues 1 to m³of the amino acid sequence in SEQ ID NO:2, where m³ is any integer inthe range of 6 to 63 (shown in SEQ ID NO:2).

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues S-1 to V-63; S-1to Y-62; S-1 to C-61; S-1 to W-60; S-1 to P-59; S-1 to G-58; S-1 toR-57; S-1 to P-56; S-1 to D-55; S-1 to E-54; S-1 to D-53; S-1 to P-52;S-1 to N-51; S-1 to R-50; S-1 to C-49; S-1 to Y-48; S-1 to S-47; S-1 toH-46; S-1 to N-45; S-1 to G-44; S-1 to A-43; S-1 to G-42; S-1 to S-41;S-1 to V-40; S-1 to P-39; S-1 to A-38; S-1 to S-37; S-1 to A-36; S-1 toL-35; S-1 to S-33; S-1 to Q-32; S-1 to A-31; S-1 to D-30; S-1 to L-29;S-1 to W-28; S-1 to N-27; S-1 to L-26; S-1 to C-25; S-1 to R-24; S-1 toL-23; S-1 to G-22; S-1 to P-21; S-1 to P-19; S-1 to S-18; S-1 to T-17;S-1 to Q-16; S-1 to D-15; S-1 to E-14; S-1 to Y-12; S-1 to L-11; S-1 toH-10; S-1 to G-9; S-1 to N-8; S-1 to D-7; and S-1 and W-6 of SEQ IDNO:2. Polynucleotides encoding these polypeptides also are provided.

Moreover, a signal sequence may be added to these C-terminal deletioncontructs. For example, amino acids Met(−)21 to Ser(+)1 of SEQ ID NO:2,amino acids Leu(−)20 to Ser(+)1 of SEQ ID NO:2, amino acids Leu(−)19 toSer(+)1 of SEQ ID NO:2, amino acids Ala(−)18 to Ser(+)1 of SEQ ID NO:2,amino acids Trp(−)17 to Ser(+)1 of SEQ ID NO:2, amino acids Val(−)16 toSer(+)1 of SEQ ID NO:2, amino acids Gln(−)15 to Ser(+)1 of SEQ ID NO:2,amino acids Ala(−)14 to Ser(+)1 of SEQ ID NO:2, amino acids Phe(−)13 toSer(+)1 of SEQ ID NO:2, amino acids Leu(−)12 to Ser(+)1 of SEQ ID NO:2,amino acids Val(−)11 to Ser(+)1 of SEQ ID NO:2, amino acids Ser(−)10 toSer(+)1 of SEQ ID NO:2, amino acids Asn(−)9 to Ser(+)1 of SEQ ID NO:2,amino acids Met(−)8 to Ser(+)1 of SEQ ID NO:2, amino acids Leu(−)7 toSer(+)1 of SEQ ID NO:2, amino acids Leu(−)6 to Ser(+)1 of SEQ ID NO:2,amino acids Ala(−)5 to Ser(+)1 of SEQ ID NO:2, amino acids Glu(−)4 toSer(+)1 of SEQ ID NO:2, amino acids Ala(−)₃ to Ser(+)1 of SEQ ID NO:2,amino acids Tyr(−)2 to Ser(+)1 of SEQ ID NO:2, and amino acids Gly(−)1to Ser(+)1 of SEQ ID NO:2 can be added or fused to the N-terminus ofeach deletion construct listed above.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini of the kringledomain of t-PALP, which may be described generally as having residuesn³–m³ of SEQ ID NO:2, where n³ and m³ are integers as described above.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete t-PALP amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 209023, wherethis portion excludes any integer of amino acid residues from 1 to about58 amino acids from the amino terminus of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209023,or any integer of amino acid residues from 1 to about 58 amino acidsfrom the carboxy terminus, or any combination of the above aminoterminal and carboxy terminal deletions, of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209023.Polynucleotides encoding all of the above deletion mutant polypeptideforms also are provided.

The present application is also directed to proteins containingpolypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to thet-PALP polypeptide sequence set forth herein m³–n³. In preferredembodiments, the application is directed to proteins containingpolypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical topolypeptides having the amino acid sequence of the specific t-PALP N-and C-terminal deletions recited herein. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the proteasedomain (Ser-64 to Ala-242) of t-PALP shown in SEQ ID NO:2, up to thealanine residue at position number 237, and polynucleotides encodingsuch polypeptides. In particular, the present invention providespolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues n⁴ to 242 of SEQ ID NO:2, where n⁴ is an integer inthe range of 64 to 237 (SEQ ID NO:2).

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues S-64 to A-242;G-65 to A-242; E-66 to A-242; A-67 to A-242; G-68 to A-242; V-69 toA-242; P-70 to A-242; E-71 to A-242; K-72 to A-242; R-73 to A-242; P-74to A-242; C-75 to A-242; E-76 to A-242; D-77 to A-242; L-78 to A-242;R-79 to A-242; C-80 to A-242; P-81 to A-242; E-82 to A-242; T-83 toA-242; T-84 to A-242; S-85 to A-242; Q-86 to A-242; A-87 to A-242; L-88to A-242; P-89 to A-242; A-90 to A-242; F-91 to A-242; T-92 to A-242;T-93 to A-242; E-94 to A-242; I-95 to A-242; Q-96 to A-242; E-97 toA-242; A-98 to A-242; S-99 to A-242; E-100 to A-242; G-101 to A-242;P-102 to A-242; G-103 to A-242; A-104 to A-242; D-105 to A-242; E-106 toA-242; V-107 to A-242; Q-108 to A-242; V-109 to A-242; F-110 to A-242;A-111 to A-242; P-112 to A-242; A-113 to A-242; N-114 to A-242; A-115 toA-242; L-116 to A-242; P-117 to A-242; A-118 to A-242; R-119 to A-242;S-120 to A-242; E-121 to A-242; A-122 to A-242; A-123 to A-242; A-124 toA-242; V-125 to A-242; Q-126 to A-242; P-127 to A-242; V-128 to A-242;I-129 to A-242; G-130 to A-242; I-131 to A-242; S-132 to A-242; Q-133 toA-242; R-134 to A-242; V-135 to A-242; R-136 to A-242; M-137 to A-242;N-138 to A-242; S-139 to A-242; K-140 to A-242; E-141 to A-242; K-142 toA-242; K-143 to A-242; D-144 to A-242; L-145 to A-242; G-146 to A-242;T-147 to A-242; L-148 to A-242; G-149 to A-242; Y-150 to A-242; V-151 toA-242; L-152 to A-242; G-153 to A-242; I-154 to A-242; T-155 to A-242;M-156 to A-242; M-157 to A-242; V-158 to A-242; I-159 to A-242; I-160 toA-242; I-161 to A-242; A-162 to A-242; I-163 to A-242; G-164 to A-242;A-165 to A-242; G-166 to A-242; I-167 to A-242; I-168 to A-242; L-169 toA-242; G-170 to A-242; Y-171 to A-242; S-172 to A-242; Y-173 to A-242;K-174 to A-242; R-175 to A-242; G-176 to A-242; K-177 to A-242; D-178 toA-242; L-179 to A-242; K-180 to A-242; E-181 to A-242; Q-182 to A-242;H-183 to A-242; D-184 to A-242; Q-185 to A-242; K-186 to A-242; V-187 toA-242; C-188 to A-242; E-189 to A-242; R-190 to A-242; E-91 to A-242;M-192 to A-242; Q-193 to A-242; R-194 to A-242; I-195 to A-242; T-196 toA-242; L-197 to A-242; P-198 to A-242; L-199 to A-242; S-200 to A-242;A-201 to A-242; F-202 to A-242; T-203 to A-242; N-204 to A-242; P-205 toA-242; T-206 to A-242; C-207 to A-242; E-208 to A-242; 1-209 to A-242;V-210 to A-242; D-211 to A-242; E-212 to A-242; K-213 to A-242; T-214 toA-242; V-215 to A-242; V-216 to A-242; V-217 to A-242; H-218 to A-242;T-219 to A-242; S-220 to A-242; Q-221 to A-242; T-222 to A-242; P-223 toA-242; V-224 to A-242; D-225 to A-242; P-226 to A-242; Q-227 to A-242;E-228 to A-242; G-229 to A-242; S-230 to A-242; T-231 to A-242; P-232 toA-242; L-233 to A-242; M-234 to A-242; G-235 to A-242; Q-236 to A-242;and A-237 to A-242 of SEQ ID NO:2. Polynucleotides encoding thesepolypeptides also are provided.

Moreover, a signal sequence may be added to these N-terminal deletioncontructs. For example, amino acids Met(−)21 to Ser(+)1 of SEQ ID NO:2,amino acids Leu(−)20 to Ser(+)1 of SEQ ID NO:2, amino acids Leu(−)19 toSer(+)1 of SEQ ID NO:2, amino acids Ala(−)18 to Ser(+)1 of SEQ ID NO:2,amino acids Trp(−)17 to Ser(+)1 of SEQ ID NO:2, amino acids Val(−)16 toSer(+)1 of SEQ ID NO:2, amino acids Gln(−)15 to Ser(+)1 of SEQ ID NO:2,amino acids Ala(−)14 to Ser(+)1 of SEQ ID NO:2, amino acids Phe(−)13 toSer(+)1 of SEQ ID NO:2, amino acids Leu(−)12 to Ser(+)1 of SEQ ID NO:2,amino acids Val(−)11 to Ser(+)1 of SEQ ID NO:2, amino acids Ser(−)10 toSer(+)1 of SEQ ID NO:2, amino acids Asn(−)9 to Ser(+)1 of SEQ ID NO:2,amino acids Met(−)8 to Ser(+)1 of SEQ ID NO:2, amino acids Leu(−)7 toSer(+)1 of SEQ ID NO:2, amino acids Leu(−)6 to Ser(+)1 of SEQ ID NO:2,amino acids Ala(−)5 to Ser(+)1 of SEQ ID NO:2, amino acids Glu(−)4 toSer(+)1 of SEQ ID NO:2, amino acids Ala(−)3 to Ser(+)1 of SEQ ID NO:2,amino acids Tyr(−)2 to Ser(+)1 of SEQ ID NO:2, and amino acids Gly(−)1to Ser(+)1 of SEQ ID NO:2 can be added to the N-terminus of eachdeletion construct listed above.

The present invention further provides polypeptides having one or moreamino acid residues deleted from the carboxy terminus of the proteasedomain (Ser-64 to Ala-242) of t-PALP (shown in SEQ ID NO:2), up to thevaline residue at position 69 of SEQ ID NO:2, and polynucleotidesencoding such polypeptides. In particular, the present inventionprovides polypeptides having the amino acid sequence of residues 64 tom⁴ of the amino acid sequence in SEQ ID NO:2, where m⁴ is any integer inthe range of 69 to 242 (shown in SEQ ID NO:2).

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues S-64 to A-242;S-64 to G-241; S-64 to P-240; S-64 to T-239; S-64 to G-238; S-64 toA-237; S-64 to Q-236; S-64 to G-235; S-64 to M-234; S-64 L-233; S-64 toP-232; S-64 to T-231; S-64 to S-230; S-64 to G-229; S-64 to E-228; S-64to Q-227; S-64 to P-226; S-64 to D-225; S-64 to V-224; S-64 to P-223;S-64 to T-222; S-64 to Q-221; S-64 to S-220; S-64 to T-219; S-64 toH-218; S-64 to V-217; S-64 to V-216; S-64 to V-215; S-64 to T-214; S-64to K-213; S-64 to E-212; S-64 to D-211; S-64 to V-210; S-64 to I-209;S-64 to E-208; S-64 to C-207; S-64 to T-206; S-64 to P-205; S-64 toN-204; S-64 to T-203; S-64 to F-202; S-64 to A-201; S-64 to S-200; S-64to L-199; S-64 to P-198; S-64 to L-197; S-64 to T-196; S-64 to I-195;S-64 to R-194; S-64 to Q-193; S-64 to M-192; S-64 to E-191; S-64 toR-190; S-64 to E-189; S-64 to C-188; S-64 to V-187; S-64 to K-186; S-64to Q-185; S-64 to D-184; S-64 to H-183; S-64 to Q-182; S-64 to E-181;S-64 to K-180; S-64 to L-179; S-64 to D-178; S-64 to K-177; S-64 toG-176; S-64 to R-175; S-64 to K-174; S-64 to Y-173; S-64 to S-172; S-64to Y-171; S-64 to G-170; S-64 to L-169; S-64 to 1-168; S-64 to 1-167;S-64 to G-166; S-64 to A-165; S-64 to G-164; S-64 to 1-163; S-64 toA-162; S-64 to 1-161; S-64 to I-160; S-64 to I-159; S-64 to V-158; S-64to M-157; S-64 to M-156; S-64 to T-155; S-64 to I-154; S-64 to G-153;S-64 to L-152; S-64 to V-151; S-64 to Y-150; S-64 to G-149; S-64 toL-148; S-64 to T-147; S-64 to G-146; S-64 to L-145; S-64 to D-144; S-64to K-143; S-64 to K-142; S-64 to E-141; S-64 to K-140; S-64 to S-139;S-64 to N-138; S-64 to M-137; S-64 to R-136; S-64 to V-135; S-64 toR-134; S-64 to Q-133; S-64 to S-132; S-64 to 1-131; S-64 to G-130; S-64to 1-129; S-64 to V-128; S-64 to P-127; S-64 to Q-126; S-64 to V-125;S-64 to A-124; S-64 to A-123; S-64 to A-122; S-64 to E-121; S-64 toS-120; S-64 to R-119; S-64 to A-118; S-64 to P-117; S-64 to L-116; S-64to A-115; S-64 to N-114; S-64 to A-113; S-64 to P-112; S-64 to A-111;S-64 to F-110; S-64 to V-109; S-64 to Q-108; S-64 to V-107; S-64 toE-106; S-64 to D-105; S-64 to A-104; S-64 to G-103; S-64 to P-102; S-64to G-101; S-64 to E-100; S-64 to S-99; S-64 to A-98; S-64 to E-97; S-64to Q-96; S-64 to I-95; S-64 to E-94; S-64 to T-93; S-64 to T-92; S-64 toF-91; S-64 to A-90; S-64 to P-89; S-64 to L-88; S-64 to A-87; S-64 toQ-86; S-64 to S-85; S-64 to T-84; S-64 to T-83; S-64 to E-82; S-64 toP-81; S-64 to C-80; S-64 to R-79; S-64 to L-78; S-64 to D-77; S-64 toE-76; S-64 to C-75; S-64 to P-74; S-64 to R-73; S-64 to K-72; S-64 toE-71; S-64 to P-70; and provided.

Moreover, a signal sequence may be added to these C-terminal deletioncontructs. For example, amino acids Met(−)21 to Ser(+)1 of SEQ ID NO:2,amino acids Leu(−)20 to Ser(+)1 of SEQ ID NO:2, amino acids Leu(−)19 toSer(+)1 of SEQ ID NO:2, amino acids Ala(−)18 to Ser(+)1 of SEQ ID NO:2,amino acids Trp(−)17 to Ser(+)1 of SEQ ID NO:2, amino acids Val(−)16 toSer(+)1 of SEQ ID NO:2, amino acids Gln(−)15 to Ser(+)1 of SEQ ID NO:2,amino acids Ala(−)14 to Ser(+)1 of SEQ ID NO:2, amino acids Phe(−)13 toSer(+)1 of SEQ ID NO:2, amino acids Leu(−)12 to Ser(+)1 of SEQ ID NO:2,amino acids Val(−)11 to Ser(+)1 of SEQ ID NO:2, amino acids Ser(−)10 toSer(+)1 of SEQ ID NO:2, amino acids Asn(−)9 to Ser(+)1 of SEQ ID NO:2,amino acids Met(−)8 to Ser(+)1 of SEQ ID NO:2, amino acids Leu(−)7 toSer(+)1 of SEQ ID NO:2, amino acids Leu(−)6 to Ser(+)1 of SEQ ID NO:2,amino acids Ala(−)5 to Ser(+)1 of SEQ ID NO:2, amino acids Glu(−)4 toSer(+)1 of SEQ ID NO:2, amino acids Ala(−)3 to Ser(+)1 of SEQ ID NO:2,amino acids Tyr(−)2 to Ser(+)1 of SEQ ID NO:2, and amino acids Gly(−)1to Ser(+)1 of SEQ ID NO:2 can be added or fused to the N-terminus ofeach deletion construct listed above.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini of the proteasedomain of t-PALP, which may be described generally as having residuesn⁴–m⁴ of SEQ ID NO:2, where n⁴ and m⁴ are integers as described above.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete t-PALP amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 209023, wherethis portion excludes any integer of amino acid residues from 1 to about178 amino acids from the amino terminus of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209023,or any integer of amino acid residues from 1 to about 178 amino acidsfrom the carboxy terminus, or any combination of the above aminoterminal and carboxy terminal deletions, of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209023.Polynucleotides encoding all of the above deletion mutant polypeptideforms also are provided.

The present application is also directed to proteins containingpolypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to thet-PALP polypeptide sequence set forth herein according to the formulam⁴–n⁴. In preferred embodiments, the application is directed to proteinscontaining polypeptides at least 90%, 95%, 96%, 97%, 98% or 99%identical to polypeptides having the amino acid sequence of the specifict-PALP N- and C-terminal deletions recited herein. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

The present application is also directed to proteins containingpolypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to thet-PALP polypeptide sequence set forth herein according to the formulaem–n, n–m, n¹–m¹, n²–m², n³–m³, and/or n⁴–m⁴. In preferred embodiments,the application is directed to proteins containing polypeptides at least90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having theamino acid sequence of the specific t-PALP N- and C-terminal deletionsrecited herein. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

In the present invention, a “polypeptide fragment” refers to an aminoacid sequence which is a portion of that contained in SEQ ID NO:2 orencoded by the cDNA contained in the deposited clone. Protein(polypeptide) fragments may be “free-standing,” or comprised within alarger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentscomprising, or alternatively consisting of, from about amino acid number(−)21–(−)1, 1–20, 21–40, 41–60, 61–80, 81–100, 102–120, 121–140,141–160, 161–180, 181–200, 201–220, or 221 to the end of the codingregion (as numbered in SEQ ID NO:2). Moreover, polypeptide fragments canbe about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150amino acids in length. In this context “about” includes the particularlyrecited ranges or values, and ranges or values larger or smaller byseveral (6, 5, 4, 3, 2, or 1) amino acids, at either extreme or at bothextremes. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Even if deletion of one or more amino acids from the N-terminus of aprotein results in modification of loss of one or more biologicalfunctions of the protein, other functional activities (e.g., biologicalactivities, ability to multimerize, ability to bind t-PALP receptor) maystill be retained. For example, the ability of shortened t-PALP muteinsto induce and/or bind to antibodies which recognize the complete ormature forms of the polypeptides generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the N-terminus. Whether a particular polypeptidelacking N-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thata t-PALP mutein with a large number of deleted N-terminal amino acidresidues may retain some biological or immunogenic activities. In fact,peptides composed of as few as six t-PALP amino acid residues may oftenevoke an immune response.

Preferred polypeptide fragments include the secreted protein as well asthe mature form, the kringle domain, and/or the protease domain. Furtherpreferred polypeptide fragments include the secreted protein, the matureform, the kringle domain, and/or the protease domain having a continuousseries of deleted residues from the amino or the carboxy terminus, orboth. For example, any number of amino acids, ranging from 1 to 85, canbe deleted from the amino terminus of either the secreted t-PALPpolypeptide or the mature form. Similarly, any number of amino acids,ranging from 1–200, can be deleted from the carboxy terminus of thesecreted t-PALP protein or mature form. Furthermore, any combination ofthe above amino and carboxy terminus deletions are preferred. Similarly,polynucleotides encoding these polypeptide fragments are also preferred.

The functional activity of t-PALP polypeptides, and fragments, variantsderivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length t-PALP polypeptide for binding toanti-t-PALP antibody, various immunoassays known in the art can be used,including but not limited to, competitive and non-competitive assaysystems using techniques such as radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

In another embodiment, where a t-PALP ligand is identified, or theability of a polypeptide fragment, variant or derivative of theinvention to multimerize is being evaluated, binding can be assayed,e.g., by means well-known in the art, such as, for example, reducing andnon-reducing gel chromatography, protein affinity chromatography, andaffinity blotting. See generally, Phizicky, E., et al., 1995, Microbiol.Rev. 59:94–123. In another embodiment, physiological correlates oft-PALP binding to its substrates (signal transduction) can be assayed.

In addition, assays described herein (see Examples) and otherwise knownin the art may routinely be applied to measure the ability of t-PALPpolypeptides and fragments, variants derivatives and analogs thereof toelicit t-PALP related biological activity (either in vitro or in vivo).Other methods will be known to the skilled artisan and are within thescope of the invention.

Among the especially preferred fragments of the invention are fragmentscharacterized by structural or functional attributes of t-PALP. Suchfragments include amino acid residues that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet andbeta-sheet-forming regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., containing four or morecontiguous amino acids having an antigenic index of greater than orequal to 1.5, as identified using the default parameters of theJameson-Wolf program) of complete (i.e., full-length) t-PALP (SEQ IDNO:2). Certain preferred regions are those set out in FIG. 3 andinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence depicted in FIGS. 1A,1B, and 1C (SEQ ID NO:2), such preferred regions include; Garnier-Robsonpredicted alpha-regions, beta-regions, turn-regions, and coil-regions;Chou-Fasman predicted alpha-regions, beta-regions, turn-regions, andcoil-regions; Kyte-Doolittle predicted hydrophilic and hydrophobicregions; Eisenberg alpha and beta amphipathic regions; Eminisurface-forming regions; and Jameson-Wolf high antigenic index regions,as predicted using the default parameters of these computer programs.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of t-PALP. Preferred embodiments of the inventionin this regard include fragments that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheetforming regions (“beta-regions”), turn and turn-forming regions(“turn-regions”), coil and coil-forming regions (“coil-regions”),hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions andhigh antigenic index regions of t-PALP.

The data representing the structural or functional attributes of t-PALPset forth in FIGS. 1A, 1B, and 1C and/or Table I, as described above,was generated using the various modules and algorithms of the DNA*STARset on default parameters. In a preferred embodiment, the data presentedin columns VIII, IX, XIII, and XIV of Table I can be used to determineregions of t-PALP which exhibit a high degree of potential forantigenicity. Regions of high antigenicity are determined from the datapresented in columns VIII, IX, XIII, and/or IV by choosing values whichrepresent regions of the polypeptide which are likely to be exposed onthe surface of the polypeptide in an environment in which antigenrecognition may occur in the process of initiation of an immuneresponse.

Certain preferred regions in these regards are set out in FIG. 3, butmay, as shown in Table I, be represented or identified by using tabularrepresentations of the data presented in FIG. 3. The DNA*STAR computeralgorithm used to generate FIG. 3 (set on the original defaultparameters) was used to present the data in FIG. 3 in a tabular format(See Table I). The tabular format of the data in FIG. 3 may be used toeasily determine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIG. 3 and in Table Iinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS. 1A,1B, and 1C. As set out in FIG. 3 and in Table 1, such preferred regionsinclude Gamier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions,Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenbergalpha- and beta-amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions and Jameson-Wolf regions of high antigenicindex.

Among highly preferred fragments in this regard are those that compriseregions of t-PALP that combine several structural features, such asseveral of the features set out above.

Other preferred polypeptide fragments are biologically active t-PALPfragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of the t-PALPpolypeptide. The biological activity of the fragments may include animproved desired activity, or a decreased undesirable activity.Polynucleotides encoding these polypeptide fragments are alsoencompassed by the invention.

However, many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a–b, where a is any integer between 1 to 2315 of SEQID NO:1, b is an integer of 15 to 2329 where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:1, and where theb is greater than or equal to a +14.

Other Mutants

In addition to terminal deletion forms of the protein discussed above,it also will be recognized by one of ordinary skill in the art that someamino acid sequences of the t-PALP polypeptide can be varied withoutsignificant effect of the structure or function of the protein. If suchdifferences in sequence are contemplated, it should be remembered thatthere will be critical areas on the protein which determine activity.

Thus, the invention further includes variations of the t-PALPpolypeptide which show substantial t-PALP polypeptide activity or whichinclude regions of t-PALP protein such as the protein portions discussedbelow. Such mutants include deletions, insertions, inversions, repeats,and type substitutions selected according to general rules known in theart so as have little effect on activity. For example, guidanceconcerning how to make phenotypically silent amino acid substitutions isprovided in Bowie, J. U. et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306–1310(1990), wherein the authors indicate that there are two main approachesfor studying the tolerance of an amino acid sequence to change. Thefirst method relies on the process of evolution, in which mutations areeither accepted or rejected by natural selection. The second approachuses genetic engineering to introduce amino acid changes at specificpositions of a cloned gene and selections or screens to identifysequences that maintain functionality.

As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described in Bowie, J. U. et al., supra, and thereferences cited therein. Typically seen as conservative substitutionsare the replacements, one for another, among the aliphatic amino acidsAla, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2, or that encoded by the deposited cDNA, may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the above form of the polypeptide, such as an IgG Fc fusionregion peptide or leader or secretory sequence or a sequence which isemployed for purification of the above form of the polypeptide or aproprotein sequence. Such fragments, derivatives and analogs are deemedto be within the scope of those skilled in the art from the teachingsherein

Thus, the t-PALP of the present invention may include one or more aminoacid substitutions, deletions or additions, either from naturalmutations or human manipulation. As indicated, changes are preferably ofa minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein (seeTable 2).

TABLE 2 Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Amino acids in the t-PALP protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081–1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro or in vitro proliferativeactivity.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331–340 (1967);Robbins et al., Diabetes 36: 838–845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307–377 (1993).

A number of mutagenesis studies have been performed on the related t-PApolypeptide. The t-PA fibrin-binding activity has been mapped to theamino-terminal finger and EGF domains (Kalyan, N. K., et al., J. Biol.Chem. 263:3971–3978; 1988). In addition, in vivo clearance rates havealso been mapped to the finger domain of t-PA (Ahern, T. J., et al., J.Biol. Chem. 265:5540–5545; 1990) Other studies by Yahara and colleagues(Thromb. and Haem. 72(6):893–899; 1994) report an in vivo clearanceactivity to be located not only in the finger domain, but also in thekringle domain of t-PA. Several mutations were identified in theprotease domain which affected t-PA protease activity (Paoni, N. F., etal., Prot. Eng. 5:259–266; 1992). Fibrinolytic activity of t-PA wasfound to be reduced by mutation of one or more highly conserved aminoacid residues in the kringle domains (Markland, W., et al., Prot. Eng.3:117–125; 1989). A key study published by Haigwood and colleagues(Prot. Eng. 2:611–620; 1989) provided a detailed analysis of the effectsof various insertion, deletion, and substitution mutations on thevarious activities of the t-PA molecule. The study determined that (1)variants with carbohydrate-depleted kringle domains possessed higherspecific activities than wild-type t-PA, (2) a cleavage site variantsubstituted at Arg275 with Gly had greatly reduced specific activity,(3) two variants substituted at Lys277 exhibited altered interactionswith plasminogen activator inhibitor (PAI)-2, (4) the variant with atruncated carboxy-terminus had reduced activity in the absence offibrin, and (5) no variants had significantly altered half-lives. Amolecular biologist skilled in the techniques of protein mutagenesiswould infer from these and other studies that, since the variousactivities of t-PA may be altered by the introduction of variousmutations into the molecule, that, by inference, it may be possible toalso target specific mutations to the t-PALP molecule in an effort toreproduce similar changes in t-PALP activities. Since t-PALP is a memberof the t-PA-related protein family, to modulate rather than completelyeliminate biological activities of t-PALP, preferably mutations are madein sequences encoding amino acids in the t-PALP conserved kringledomain, i.e., in positions 4 to 63 of SEQ ID NO:2, more preferably inresidues within this region which are not conserved in all members ofthe t-PA-related protein family. Similarly, preferable mutations aremade in sequences encoding amino acids in the t-PALP conserved proteasedomain, i.e., in positions 64 to 242 of SEQ ID NO:2, more preferably inresidues within this region which are not conserved in all members ofthe t-PA-related protein family. Also forming part of the presentinvention are isolated polynucleotides comprising nucleic acid sequenceswhich encode the above t-PALP mutants.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of the t-PALP polypeptide can besubstantially purified by the one-step method described by Smith andJohnson (Gene 67:31–40; 1988). Polypeptides of the invention also can bepurified from natural or recombinant sources using anti-t-PALPantibodies of the invention in methods which are well known in the artof protein purification.

The invention further provides an isolated t-PALP polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) theamino acid sequence of the full-length t-PALP polypeptide having thecomplete amino acid sequence shown in SEQ ID NO:2 excepting theN-terminal methionine (i.e., positions −20 to 242 of SEQ ID NO:2) or thecomplete amino acid sequence excepting the N-terminal methionine encodedby the cDNA clone contained in the ATCC Deposit No. 209023; (b) theamino acid sequence comprising the predicted mature form of the t-PALPpolypeptide having the amino acid sequence at positions 1 to 242 in SEQID NO:2 or as encoded by the cDNA clone contained in the ATCC DepositNo. 209023; (c) the amino acid sequence comprising the predicted kringledomain of the t-PALP polypeptide having the amino acid sequence atpositions 4 to 63 in SEQ ID NO:2 or as encoded by the cDNA clonecontained in the ATCC Deposit No. 209023; and (d) the amino acidsequence comprising the predicted protease domain of the t-PALPpolypeptide having the amino acid sequence at positions 64 to 242 in SEQID NO:2 or as encoded by the cDNA clone contained in the ATCC DepositNo. 209023. The polypeptides of the present invention also includepolypeptides having an amino acid sequence at least 80% identical (or20% different), more preferably at least 90% identical (or 10%different), and still more preferably 95%, 96%, 97%, 98% or 99%identical to (or 5%, 4%, 3%, 2% or 1% different from) those described in(a), (b), (c) or (d) above, as well as polypeptides having an amino acidsequence with at least 90% similarity, and more preferably at least 95%similarity, to those above.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 95% similarity,and still more preferably at least 96%, 97%, 98% or 99% similarity tothose described above. The polypeptides of the invention also comprisethose which are at least 80% identical, more preferably at least 90% or95% identical, still more preferably at least 96%, 97%, 98% or 99%identical to the polypeptide encoded by the deposited cDNA or to thepolypeptide of SEQ ID NO:2, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2:482–489, 1981) tofind the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a t-PALPpolypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the t-PALP polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to (or 5% different from) a reference amino acidsequence, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in SEQ ID NO:2, the amino acid sequence encoded bythe deposited plasmid DNA HMS1B42 (ATCC Accession No. 209023), orfragments thereof, or, for instance, to the amino acid sequence shown inFIG. 1 (SEQ ID NO:2), the amino acid sequence encoded by the depositedcDNA clone HMS1B42 (ATCC Accession No. 209023), or fragments thereof,can be determined conventionally using known computer programs such theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the reference aminoacid sequence and that gaps in homology of up to 5% of the total numberof amino acid residues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237–245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N- or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N- and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence. For example, a 90 aminoacid residue subject sequence is aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N- or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

A further embodiment of the invention relates to a peptide orpolypeptide which comprises the amino acid sequence of a t-PALPpolypeptide having an amino acid sequence which contains at least oneconservative amino acid substitution, but not more than 50 conservativeamino acid substitutions, even more preferably, not more than 40conservative amino acid substitutions, still more preferably, not morethan 30 conservative amino acid substitutions, and still even morepreferably, not more than 20 conservative amino acid substitutions. Ofcourse, in order of ever-increasing preference, it is highly preferablefor a peptide or polypeptide to have an amino acid sequence whichcomprises the amino acid sequence of a t-PALP polypeptide, whichcontains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1conservative amino acid substitutions.

The polypeptide of the present invention could be used as a molecularweight marker on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art.

As described in detail below, the polypeptides of the present inventioncan also be used to raise polyclonal and monoclonal antibodies, whichare useful in assays for detecting t-PALP protein expression asdescribed below or as agonists and antagonists capable of enhancing orinhibiting t-PALP protein function. Further, such polypeptides can beused in the yeast two-hybrid system to “capture” t-PALP protein bindingproteins which are also candidate agonists and antagonists according tothe present invention. The yeast two hybrid system is described inFields and Song, Nature 340:245–246 (1989).

Epitope-Bearing Portions

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998–4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Leamer, R. A. (1983) “Antibodies that react withpredetermined sites on proteins,” Science, 219:660–666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals. Antigenic epitope-bearing peptides and polypeptidesof the invention are therefore useful to raise antibodies, includingmonoclonal antibodies, that bind specifically to a polypeptide of theinvention. See, for instance, Wilson et al., Cell 37:767–778 (1984) at777.

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between about 15 to about 30 amino acidscontained within the amino acid sequence of a polypeptide of theinvention. Non-limiting examples of antigenic polypeptides or peptidesthat can be used to generate t-PALP-specific antibodies include: apolypeptide comprising amino acid residues from about Ser-1 to aboutHis-10 in SEQ ID NO:2; about Glu-14 to about Leu-23 in SEQ ID NO:2;about Arg-50 to about Trp-60 in SEQ ID NO:2; about Pro-70 to aboutGln-86 in SEQ ID NO:2; about Ala-98 to about Val-107 in SEQ ID NO:2;about Leu-117 to about Gln-126 in SEQ ID NO:2; about Arg-134 to aboutGly-146 in SEQ ID NO:2; about Ser-172 to about Gln-182 in SEQ ID NO:2;about Gln-185 to about Arg-194 in SEQ ID NO:2; about Thr-206 to aboutVal-216 in SEQ ID NO:2; and about Thr-222 to about Thr-231 in SEQ IDNO:2; These polypeptide fragments have been determined to bear antigenicepitopes of the t-PALP protein by the analysis of the Jameson-Wolfantigenic index, as shown in FIG. 3, above.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. See, e.g., Houghten, R. A. (1985)“General method for the rapid solid-phase synthesis of large numbers ofpeptides: specificity of antigen-antibody interaction at the level ofindividual amino acids.” Proc. Natl. Acad. Sci. USA 82:5131–5135; this“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. etal., Proc. Natl. Acad. Sci. USA 82:910–914; and Bittle, F. J. et al., J.Gen. Virol. 66:2347–2354 (1985). Immunogenic epitope-bearing peptides ofthe invention, i.e., those parts of a protein that elicit an antibodyresponse when the whole protein is the immunogen, are identifiedaccording to methods known in the art. See, for instance, Geysen et al.,supra. Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describesa general method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C1–C7-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

The present invention is also directed to polypeptide fragmentscomprising, or alternatively consisting of, an epitope of thepolypeptide sequence shown in SEQ ID NO:2, or the polypeptide sequenceencoded by the cDNA contained in a deposited clone. Polynucleotidesencoding these epitopes (such as, for example, the sequence disclosed inSEQ ID NO:1) are also encompassed by the invention, as is the nucleotidesequences of the complementary strand of the polynucleotides encodingthese epitopes. And polynucleotides which hybridize to the complementarystrand under stringent hybridization conditions or lower stringencyconditions.

In the present invention, “epitopes” refer to polypeptide fragmentshaving antigenic or immunogenic activity in an animal, especially in ahuman. A preferred embodiment of the present invention relates to apolypeptide fragment comprising an epitope, as well as thepolynucleotide encoding this fragment. A region of a protein molecule towhich an antibody can bind is defined as an “antigenic epitope.” Incontrast, an “immunogenic epitope” is defined as a part of a proteinthat elicits an antibody response. (See, for instance, Geysen et al.,Proc. Natl. Acad. Sci. USA 81:3998–4002 (1983).)

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA82:5131–5135 (1985) further described in U.S. Pat. No. 4,631,211.)

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, and most preferably between about 15 to about 30 aminoacids. Preferred polypeptides comprising immunogenic or antigenicepitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenicepitopes are useful, for example, to raise antibodies, includingmonoclonal antibodies, that specifically bind the epitope. (See, forinstance, Wilson et al., Cell 37:767–778 (1984); Sutcliffe et al.,Science 219:660–666 (1983).)

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910–914; and Bittle et al., J. Gen. Virol.66:2347–2354 (1985).) A preferred immunogenic epitope includes thesecreted protein. The immunogenic epitopes may be presented togetherwith a carrier protein, such as an albumin, to an animal system (such asrabbit or mouse) or, if it is long enough (at least about 25 aminoacids), without a carrier. However, immunogenic epitopes comprising asfew as 8 to 10 amino acids have been shown to be sufficient to raiseantibodies capable of binding to, at the very least, linear epitopes ina denatured polypeptide (e.g., in Western blotting.)

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347–2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingof the peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such as-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μgs of peptide or carrier protein and Freund's adjuvant.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

As one of skill in the art will appreciate, and discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to heterologous polypeptide sequences.For example, the polypeptides of the present invention may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, any combination thereof including both entiredomains and portions thereof) resulting in chimeric polypeptides. Thesefusion proteins facilitate purification, and show an increased half-lifein vivo. This has been shown, e.g., for chimeric proteins consisting ofthe first two domains of the human CD4-polypeptide and various domainsof the constant regions of the heavy or light chains of mammalianimmunoglobulins. See, e.g., EPA 0,394,827; Traunecker et al., Nature,331:84–86 (1988). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG portion can also be more efficient in bindingand neutralizing other molecules than monomeric polypeptides orfragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958–3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag to aid indetection and purification of the expressed polypeptide.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptidescorresponding to SEQ ID NO:2 thereby effectively generating agonists andantagonists of the polypeptides. See, generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten,P. A., et al., Curr. Opinion Biotechnol. 8:724–33 (1997); Harayama, S.,Trends Biotechnol. 16(2):76–82 (1998); Hansson, L. O., et al., J. Mol.Biol. 287:265–76 (1999); and Lorenzo, M. M. and Blasco, R.,Biotechniques 24(2):308–13 (1998) (each of these patents andpublications are hereby incorporated by reference). In one embodiment,alteration of polynucleotides corresponding to SEQ ID NO:1 andcorresponding polypeptides may be achieved by DNA shuffling. DNAshuffling involves the assembly of two or more DNA segments into adesired molecule corresponding to SEQ ID NO:1 polynucleotides of theinvention by homologous, or site-specific, recombination. In anotherembodiment, polynucleotides corresponding to SEQ ID NO:1 andcorresponding polypeptides may be altered by being subjected to randommutagenesis by error-prone PCR, random nucleotide insertion or othermethods prior to recombination. In another embodiment, one or morecomponents, motifs, sections, parts, domains, fragments, etc., of codingpolynucleotide corresponding to SEQ ID NO:1, or the polypeptide encodedthereby may be recombined with one or more components, motifs, sections,parts, domains, fragments, etc. of one or more heterologous molecules.

Fusion Proteins

As one of skill in the art will appreciate, t-PALP polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EP A 394,827; Traunecker et al., Nature 331:84–86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric t-PALP protein orprotein fragment alone (Fountoulakis et al., J. Biochem. 270:3958–3964(1995)).

Any t-PALP polypeptide can be used to generate fusion proteins. Forexample, the t-PALP polypeptide, when fused to a second protein, can beused as an antigenic tag. Antibodies raised against the t-PALPpolypeptide can be used to indirectly detect the second protein bybinding to the t-PALP. Moreover, because secreted proteins targetcellular locations based on trafficking signals, the t-PALP polypeptidescan be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused to t-PALP polypeptides include notonly heterologous signal sequences, but also other heterologousfunctional regions. The fusion does not necessarily need to be direct,but may occur through linker sequences.

In certain preferred embodiments, t-PALP proteins of the inventioncomprise fusion proteins wherein the t-PALP polypeptides are thosedescribed above as n¹–m¹, n²–m², n³–m³, n⁴–m⁴. In preferred embodiments,the application is directed to nucleic acid molecules at least 90%, 95%,96%, 97%, 98% or 99% identical to the nucleic acid sequences encodingpolypeptides having the amino acid sequence of the specific N- andC-terminal deletions recited herein. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the t-PALP polypeptide. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the t-PALP polypeptide to improve stability andpersistence during purification from the host cell or subsequenthandling and storage. Also, peptide moieties may be added to the t-PALPpolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the t-PALP polypeptide. The addition ofpeptide moieties to facilitate handling of polypeptides are familiar androutine techniques in the art.

As one of skill in the art will appreciate, polypeptides of the presentinvention and the epitope-bearing fragments thereof described above, canbe combined with heterologous polypeptide sequences. For example, thepolypeptides of the present invention may be fused with heterologouspolypeptide sequences, for example, the polypeptides of the presentinvention may be fused with parts of the constant domain ofimmunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3,and any combination thereof, including both entire domains and portionsthereof), resulting in chimeric polypeptides. These fusion proteinsfacilitate purification and show an increased half-life in vivo. Onereported example describes chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins. (EP A394,827; Traunecker et al., Nature 331:84–86 (1988).) Fusion proteinshaving disulfide-linked dimeric structures (due to the IgG) can also bemore efficient in binding and neutralizing other molecules, than themonomeric secreted protein or protein fragment alone. (Fountoulakis etal., J. Biochem. 270:3958–3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52–58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459–9471 (1995).)

Moreover, the t-PALP polypeptides can be fused to marker sequences, suchas a peptide which facilitates purification of t-PALP. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821–824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984).)

Thus, any of these above fusions can be engineered using the t-PALPpolynucleotides or the polypeptides.

Antibodies

-   -   t-PALP-protein specific antibodies for use in the present        invention can be raised against the intact t-PALP protein or an        antigenic polypeptide fragment thereof, which may be presented        together with a carrier protein, such as an albumin, to an        animal system (such as rabbit or mouse) or, if it is long enough        (at least about 25 amino acids), without a carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to t-PALP protein. Fab and F(ab′)2 fragments lackthe Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl et al., J Nucl. Med. 24:316–325 (1983)). Thus, thesefragments are preferred.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the t-PALP protein oran antigenic fragment thereof can be administered to an animal in orderto induce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of t-PALP protein is prepared andpurified to render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or t-PALP protein binding fragments thereof).Such monoclonal antibodies can be prepared using hybridoma technology(Kohler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol.6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerlinget al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,(1981) pp. 563–681). In general, such procedures involve immunizing ananimal (preferably a mouse) with a t-PALP protein antigen or, morepreferably, with a t-PALP protein-expressing cell. Suitable cells can berecognized by their capacity to bind anti-t-PALP protein antibody. Suchcells may be cultured in any suitable tissue culture medium; however, itis preferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 g/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin. Thesplenocytes of such mice are extracted and fused with a suitable myelomacell line. Any suitable myeloma cell line may be employed in accordancewith the present invention; however, it is preferable to employ theparent myeloma cell line (SP2O), available from the American TypeCulture Collection, Rockville, Md. After fusion, the resulting hybridomacells are selectively maintained in HAT medium, and then cloned bylimiting dilution as described by Wands et al. (Gastroenterology80:225–232 (1981)). The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding the t-PALP protein antigen.

Alternatively, additional antibodies capable of binding to the t-PALPprotein antigen may be produced in a two-step procedure through the useof anti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and that, therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, t-PALP-protein specific antibodies are used toimmunize an animal, preferably a mouse. The splenocytes of such ananimal are then used to produce hybridoma cells, and the hybridoma cellsare screened to identify clones which produce an antibody whose abilityto bind to the t-PALP protein-specific antibody can be blocked by thet-PALP protein antigen. Such antibodies comprise anti-idiotypicantibodies to the t-PALP protein-specific antibody and can be used toimmunize an animal to induce formation of further t-PALPprotein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, t-PALPprotein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

For in vivo use of anti-t-PALP in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which specifically bind the polypeptides of the presentinvention. The antibodies of the present invention include IgG(including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2),IgD, IgE, or IgM, and IgY. As used herein, the term “antibody” (Ab) ismeant to include whole antibodies, including single-chain wholeantibodies, and antigen-binding fragments thereof. Most preferably theantibodies are human antigen binding antibody fragments of the presentinvention and include, but are not limited to, Fab, Fab′ and F(ab′)2,Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linkedFvs (sdFv) and fragments comprising either a V_(L) or V_(H) domain. Theantibodies may be from any animal origin including birds and mammals.Preferably, the antibodies are human, murine, rabbit, goat, guinea pig,camel, horse, or chicken.

Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentire or partial of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are any combinations of variableregion(s) and hinge region, CH1, CH2, and CH3 domains. The presentinvention further includes monoclonal, polyclonal, chimeric, humanized,and human monoclonal and human polyclonal antibodies which specificallybind the polypeptides of the present invention. The present inventionfurther includes antibodies which are anti-idiotypic to the antibodiesof the present invention.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60–69 (1991); U.S. Pat. Nos. 5,573,920, 4,474,893,5,601,819, 4,714,681, 4,925,648; Kostelny et al., J. Immunol.148:1547–1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which are recognized or specifically bound by the antibody.The epitope(s) or polypeptide portion(s) may be specified as describedherein, e.g., by N-terminal and C-terminal positions, by size incontiguous amino acid residues, or listed in the Tables and Figures.Antibodies which specifically bind any epitope or polypeptide of thepresent invention may also be excluded. Therefore, the present inventionincludes antibodies that specifically bind polypeptides of the presentinvention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of the polypeptides of the presentinvention are included. Antibodies that do not bind polypeptides withless than 95%, less than 90%, less than 85%, less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, andless than 50% identity (as calculated using methods known in the art anddescribed herein) to a polypeptide of the present invention are alsoincluded in the present invention. Further included in the presentinvention are antibodies which only bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M,5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. For example, the antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of the polypeptides of the present invention in biologicalsamples. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated byreference in the entirety).

The antibodies of the present invention may be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 0 396 387.

The antibodies of the present invention may be prepared by any suitablemethod known in the art. For example, a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. The term “monoclonal antibody” is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced. Monoclonal antibodies can be prepared using a wide varietyof techniques known in the art including the use of hybridoma,recombinant, and phage display technology.

Hybridoma techniques include those known in the art and taught in Harlowet al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: MONOCLONAL ANTIBODIES ANDT-CELL HYBRIDOMAS 563–681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). Fab and F(ab′)2fragments may be produced by proteolytic cleavage, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments).

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA and phage display technologyor through synthetic chemistry using methods known in the art. Forexample, the antibodies of the present invention can be prepared usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface of aphage particle which carries polynucleotide sequences encoding them.Phage with a desired binding property are selected from a repertoire orcombinatorial antibody library (e.g. human or murine) by selectingdirectly with antigen, typically antigen bound or captured to a solidsurface or bead. Phage used in these methods are typically filamentousphage including fd and M13 with Fab, Fv or disulfide stabilized Fvantibody domains recombinantly fused to either the phage gene III orgene VIII protein. Examples of phage display methods that can be used tomake the antibodies of the present invention include those disclosed inBrinkman et al., J. Immunol. Methods 182:41–50 (1995); Ames et al., J.Immunol. Methods 184:177–186 (1995); Kettleborough et al., Eur. J.Immunol. 24:952–958 (1994); Persic et al., Gene 187 9–18 (1997); Burtonet al., Advances in Immunology 57:191–280 (1994); PCT/GB91/01134; WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426, 5,223,409,5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698,5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743 (saidreferences incorporated by reference in their entireties).

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)2 fragments can also be employed using methods known in the artsuch as those disclosed in WO 92/22324; Mullinax et al., BioTechniques12(6):864–869 (1992); and Sawai et al., AJRI 34:26–34 (1995); and Betteret al., Science 240:1041–1043 (1988) (said references incorporated byreference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46–88 (1991); Shu,L. et al., PNAS 90:7995–7999 (1993); and Skerra et al., Science240:1038–1040 (1988). For some uses, including in vivo use of antibodiesin humans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.Immunol. Methods 125:191–202; and U.S. Pat. No. 5,807,715. Antibodiescan be humanized using a variety of techniques including CDR-grafting(EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and 5,585,089),veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E. A.,Molecular Immunology 28(4/5):489–498 (1991); Studnicka et al., ProteinEngineering 7(6):805–814 (1994); Roguska. et al., PNAS 91:969–973(1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Human antibodiescan be made by a variety of methods known in the art including phagedisplay methods described above. See also, U.S. Pat. Nos. 4,444,887,4,716,111, 5,545,806, and 5,814,318; and WO 98/46645, WO 98/50433, WO98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 (saidreferences incorporated by reference in their entireties).

Further included in the present invention are antibodies recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugations) to a polypeptide of the present invention.The antibodies may be specific for antigens other than polypeptides ofthe present invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal. supra and WO 93/21232; EP 0 439 095; Naramura et al., Immunol. Lett.39:91–99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS89:1428–1432 (1992); Fell et al., J. Immunol. 146:2446–2452(1991) (saidreferences incorporated by reference in their entireties).

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the hinge region, CH1 domain, CH2domain, and CH3 domain or any combination of whole domains or portionsthereof. The polypeptides of the present invention may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. The polypeptides may also be fused or conjugated to theabove antibody portions to form multimers. For example, Fc portionsfused to the polypeptides of the present invention can form dimersthrough disulfide bonding between the Fc portions. Higher multimericforms can be made by fusing the polypeptides to portions of IgA and IgM.Methods for fusing or conjugating the polypeptides of the presentinvention to antibody portions are known in the art. See e.g., U.S. Pat.Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946;EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi et al.,PNAS 88:10535–10539 (1991); Zheng et al., J. Immunol. 154:5590–5600(1995); and Vil et al., PNAS 89:11337–11341(1992) (said referencesincorporated by reference in their entireties).

The invention further relates to antibodies which act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Included are both receptor-specificantibodies and ligand-specific antibodies. Included arereceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. Also included are receptor-specific antibodies which both preventligand binding and receptor activation. Likewise, included areneutralizing antibodies which bind the ligand and prevent binding of theligand to the receptor, as well as antibodies which bind the ligand,thereby preventing receptor activation, but do not prevent the ligandfrom binding the receptor. Further included are antibodies whichactivate the receptor. These antibodies may act as agonists for eitherall or less than all of the biological activities affected byligand-mediated receptor activation. The antibodies may be specified asagonists or antagonists for biological activities comprising specificactivities disclosed herein. The above antibody agonists can be madeusing methods known in the art. See e.g., WO 96/40281; U.S. Pat. No.5,811,097; Deng et al., Blood 92(6):1981–1988 (1998); Chen, et al.,Cancer Res. 58(16):3668–3678 (1998); Harrop et al., J. Immunol.161(4):1786–1794 (1998); Zhu et al., Cancer Res. 58(15):3209–3214(1998); Yoon, et al., J. Immunol. 160(7):3170–3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237–247 (1998); Pitard et al., J. Immunol.Methods 205(2):177–190 (1997); Liautard et al., Cytokinde 9(4):233–241(1997); Carlson et al., J. Biol. Chem. 272(17):11295–11301 (1997);Taryman et al., Neuron 14(4):755–762 (1995); Muller et al., Structure6(9):1153–1167 (1998); Bartunek et al., Cytokine 8(1):14–20 (1996) (saidreferences incorporated by reference in their entireties).

As discussed above, antibodies to the polypeptides of the invention can,in turn, be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437–444;(1989) and Nissinoff, J. Immunol. 147(8):2429–2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention toligand can be used to generate anti-idiotypes that “mimic” thepolypeptide mutimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

The invention further relates to a diagnostic kit for use in screeningserum containing antibodies specific against proliferative and/orcancerous polynucleotides and polypeptides. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope whichis specifically immunoreactive with at least one anti-polypeptideantigen antibody. Such a kit also includes means for detecting thebinding of said antibody to the antigen. In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedpolypeptide antigen. The polypeptide antigen of the kit may also beattached to a solid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labelledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labelled antibody.

The invention further includes a method of detecting proliferativeand/or cancerous disorders and conditions in a test subject. Thisdetection method includes reacting serum from a test subject (e.g. onein which proliferative and/or cancerous cells or tissues may be present)with a substantially isolated polypeptide and/or polynucleotide antigen,and examining the antigen for the presence of bound antibody. In aspecific embodiment, the method includes a polypeptide antigen attachedto a solid support, and the serum is reacted with the support.Subsequently, the support is reacted with a reporter labelled anti-humanantibody. The solid support is then examined for the presence ofreporter-labelled antibody.

Additionally, the invention includes a proliferative condition vaccinecomposition. The composition includes a substantially isolatedpolypeptide and/or polynucleotide antigen, where the antigen includes anepitope which is specifically immunoreactive with at least antibodyspecific for the epitope. The peptide and/or polynucleotide antigen maybe produced according to methods known in the art, including recombinantexpression or chemical synthesis. The peptide antigen is preferablypresent in a pharmacologically effective dose in a pharmaceuticallyacceptable carrier.

Further, the invention includes a monoclonal antibody that isspecifically immunoreactive with polypeptide and/or polynucleotideepitopes. The invention includes a substantially isolated preparation ofpolyclonal antibodies specifically immunoreactive with polynucleotidesand/or polypeptides of the present invention. In a more specificembodiment, such polyclonal antibodies are prepared by affinitychromatography, in addition to, other methods known in the art.

In another embodiment, the invention includes a method for producingantibodies to polypeptide and/or polynucleotide antigens. The methodincludes administering to a test subject a substantially isolatedpolypeptide and/or polynucleotide antigen, where the antigen includes anepitope which is specifically immunoreactive with at least oneanti-polypeptide and/or polynucleotide antibody. The antigen isadministered in an amount sufficient to produce an immune response inthe subject.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labelled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labelled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labelled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labelled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric or colorimetric substrate (Sigma, St. Louis,Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labelled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Circulatory System-Related Disorders

Diagnosis

The present inventors have discovered that t-PALP is expressed inactivated monocytes. For a number of circulatory system-relateddisorders, substantially altered (increased or decreased) levels oft-PALP gene expression can be detected in circulatory system tissue orother cells or bodily fluids (e.g., sera, plasma, urine, synovial fluidor spinal fluid) taken from an individual having such a disorder,relative to a “standard” t-PALP gene expression level, that is, thet-PALP expression level in circulatory system tissues or bodily fluidsfrom an individual not having the circulatory system disorder. Thus, theinvention provides a diagnostic method useful during diagnosis of acirculatory system disorder, which involves measuring the expressionlevel of the gene encoding the t-PALP protein in circulatory systemtissue or other cells or body fluid from an individual and comparing themeasured gene expression level with a standard t-PALP gene expressionlevel, whereby an increase or decrease in the gene expression levelcompared to the standard is indicative of an circulatory systemdisorder.

In particular, it is believed that certain tissues in mammals withcancers of the circulatory system express significantly reduced levelsof the t-PALP protein and mRNA encoding the t-PALP protein when comparedto a corresponding “standard” level. Further, it is believed thataltered levels of the t-PALP protein can be detected in certain bodyfluids (e.g., sera, plasma, urine, and spinal fluid) from mammals withsuch a cancer when compared to sera from mammals of the same species nothaving the cancer.

Thus, the invention provides a diagnostic method useful during diagnosisof a circulatory system disorder, including cancers of this system,which involves measuring the expression level of the gene encoding thet-PALP protein in the circulatory system tissue or other cells or bodyfluid from an individual and comparing the measured gene expressionlevel with a standard t-PALP gene expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a circulatory system disorder.

Where a diagnosis of a disorder in the circulatory system includingdiagnosis of a cancer has already been made according to conventionalmethods, the present invention is useful as a prognostic indicator,whereby patients exhibiting enhanced or depressed t-PALP gene expressionwill experience a worse clinical outcome relative to patients expressingthe gene at a level nearer the standard level.

By “assaying the expression level of the gene encoding the t-PALPprotein” is intended qualitatively or quantitatively measuring orestimating the level of the t-PALP protein or the level of the mRNAencoding the t-PALP protein in a first biological sample either directly(e.g., by determining or estimating absolute protein level or mRNAlevel) or relatively (e.g., by comparing to the t-PALP protein level ormRNA level in a second biological sample). Preferably, the t-PALPprotein level or mRNA level in the first biological sample is measuredor estimated and compared to a standard t-PALP protein level or mRNAlevel, the standard being taken from a second biological sample obtainedfrom an individual not having the disorder or being determined byaveraging levels from a population of individuals not having a disorderof the circulatory system. As will be appreciated in the art, once astandard t-PALP protein level or mRNA level is known, it can be usedrepeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains t-PALP protein or mRNA. As indicated, biological samplesinclude body fluids (such as sera, plasma, urine, synovial fluid andspinal fluid) which contain free t-PALP protein, circulatory systemtissue, and other tissue sources found to express complete or maturet-PALP or a t-PALP receptor. Methods for obtaining tissue biopsies andbody fluids from mammals are well known in the art. Where the biologicalsample is to include mRNA, a tissue biopsy is the preferred source.

The present invention is useful for diagnosis or treatment of variouscirculatory system-related disorders in mammals, preferably humans. Suchdisorders include any disregulation of circulatory cell functionincluding, but not limited to, diseases related to thrombosis, which ischaracterized by hypercoagulation of blood cells. t-PALP may be employedto prevent proximal extension of deep-venous thrombosis or therecurrence of pulmonary embolisms, which are characterized by lodging ofa blood clot in a pulmonary artery with subsequent obstruction of bloodsupply to the lung parenchyma. t-PALP may also be employed to helpprevent the recurrence of cerebral or other systemic embolisms. Theenzyme of the present invention may also be used to treat high riskpatients, such as those who have congestive heart failure, acutemyocardial infarction or cardiomyopathy to prevent the development ofdeep-vein thrombosis or pulmonary embolism. t-PALP may also be employedas a long-term therapy for the occasional patient who has recurrentthrombosis or embolism while on the drug Warfarin.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156–159 (1987). Levels ofmRNA encoding the t-PALP protein are then assayed using any appropriatemethod. These include Northern blot analysis, S1 nuclease mapping, thepolymerase chain reaction (PCR), reverse transcription in combinationwith the polymerase chain reaction (RT-PCR), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

Assaying t-PALP protein levels in a biological sample can occur usingantibody-based techniques. For example, t-PALP protein expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976–985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087–3096 (1987)). Other antibody-basedmethods useful for detecting t-PALP protein gene expression includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA). Suitable antibody assay labels are known inthe art and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹²In), and technetium (^(99m)Tc), and fluorescentlabels, such as fluorescein and rhodamine, and biotin.

In addition to assaying t-PALP protein levels in a biological sampleobtained from an individual, t-PALP protein can also be detected in vivoby imaging. Antibody labels or markers for in vivo imaging of t-PALPprotein include those detectable by X-radiography, NMR or ESR. ForX-radiography, suitable labels include radioisotopes such as barium orcesium, which emit detectable radiation but are not overtly harmful tothe subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

A t-PALP protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for immune systemdisorder. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain t-PALP protein. Invivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

Treatment

As noted above, t-PALP polynucleotides and polypeptides are useful fordiagnosis of conditions involving abnormally high or low expression oft-PALP activities. Given the cells and tissues where t-PALP is expressedas well as the activities modulated by t-PALP, it is readily apparentthat a substantially altered (increased or decreased) level ofexpression of t-PALP in an individual compared to the standard or“normal” level produces pathological conditions related to the bodilysystem(s) in which t-PALP is expressed and/or is active.

It will also be appreciated by one of ordinary skill that, since thet-PALP protein of the invention is related to t-PA the mature secretedform of the protein may be released in soluble form from the cells whichexpress the t-PALP by proteolytic cleavage. Therefore, when t-PALPmature form is added from an exogenous source to cells, tissues or thebody of an individual, the protein will exert its physiologicalactivities on its target cells of that individual.

Therefore, it will be appreciated that conditions caused by a decreasein the standard or normal level of t-PALP activity in an individual,particularly disorders of the circulatory system, can be treated byadministration of t-PALP polypeptide (in the form of the mature,secreted protein). Thus, the invention also provides a method oftreatment of an individual in need of an increased level of t-PALPactivity comprising administering to such an individual a pharmaceuticalcomposition comprising an amount of an isolated t-PALP polypeptide ofthe invention, particularly a mature form of the t-PALP protein of theinvention, effective to increase the t-PALP activity level in such anindividual.

t-PALP may also be employed in combinations, compositions, and methodsfor treating thrombic disease. For example, the enzyme of the presentinvention may be combined with a thrombolytic agent to work in acomplementary fashion to dissolve blood clots, resulting in decreasedreperfusion times and increased reocclusion times in patients. Thethrombolytic agent dissolves the clot while t-PALP prevents thrombinfrom regenerating the clot. This combination allows the administrationof a thrombolytic agent at a considerably lower dosage than if givenalone, therefore, allowing the prevention of undesirable side-effectsassociated with the use of a high level of thrombolytic agent, forexample, bleeding complications.

Formulations

The t-PALP polypeptide composition will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of the individual patient (especially the sideeffects of treatment with t-PALP polypeptide alone), the site ofdelivery of the t-PALP polypeptide composition, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” of t-PALP polypeptide forpurposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount oft-PALP polypeptide administered parenterally per dose will be in therange of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the t-PALP polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1–4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

Pharmaceutical compositions containing the t-PALP of the invention maybe administered orally, rectally, parenterally, intracistemally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

The t-PALP polypeptide is also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. etal., Biopolymers 22:547–556 (1983)), poly (2-hydroxyethyl methacrylate)(R. Langer et al., J. Biomed. Mater. Res. 15:167–277 (1981), and R.Langer, Chem. Tech. 12:98–105 (1982)), ethylene vinyl acetate (R. Langeret al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release t-PALP polypeptide compositions also includeliposomally entrapped t-PALP polypeptide. Liposomes containing t-PALPpolypeptide are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688–3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. (USA) 77:4030–4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200–800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalt-PALP polypeptide therapy.

For parenteral administration, in one embodiment, the t-PALP polypeptideis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the t-PALPpolypeptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The t-PALP polypeptide is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1–10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of t-PALP polypeptide salts.

t-PALP polypeptide to be used for therapeutic administration must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic t-PALPpolypeptide compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

t-PALP polypeptide ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous t-PALP polypeptide solution, andthe resulting mixture is lyophilized. The infusion solution is preparedby reconstituting the lyophilized t-PALP polypeptide usingbacteriostatic Water-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

Agonists and Antagonists—Assays and Molecules

The invention also provides a method of screening compounds to identifythose which enhance or block the action of t-PALP on cells, such as itsinteraction with t-PALP-binding molecules. An agonist is a compoundwhich increases the natural biological functions of t-PALP or whichfunctions in a manner similar to t-PALP, while antagonists decrease oreliminate such functions.

In another aspect of this embodiment the invention provides a method foridentifying a protein which binds specifically to a t-PALP polypeptide.For example, the t-PALP polypeptide may be bound to a solid support sothat binding molecules solubilized from cells are bound to the columnand then eluted and characterized according to routine methods.

In the assay of the invention for agonists or antagonists, a cellularcompartment, such as a membrane or a preparation thereof, may beprepared from a cell that expresses a molecule that binds t-PALP. Thepreparation is incubated with labeled t-PALP in the absence or thepresence of a candidate molecule which may be a t-PALP agonist orantagonist. The ability of the candidate molecule to bind the bindingmolecule is reflected in decreased binding of the labeled ligand.Molecules which bind gratuitously, i.e., without inducing the effects oft-PALP on binding the t-PALP binding molecule, are most likely to begood antagonists. Molecules that bind well and elicit effects that arethe same as or closely related to t-PALP are agonists.

t-PALP-like effects of potential agonists and antagonists may bymeasured, for instance, by determining activity of a second messengersystem following interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that oft-PALP or molecules that elicit the same effects as t-PALP. Secondmessenger systems that may be useful in this regard include but are notlimited to AMP guanylate cyclase, ion channel or phosphoinositidehydrolysis second messenger systems.

Another example of an assay for t-PALP antagonists is a competitiveassay that combines t-PALP and a potential antagonist with recombinantt-PALP receptor molecules under appropriate conditions for a competitiveinhibition assay. t-PALP can be labeled, such as by radioactivity, suchthat the number of t-PALP molecules bound to a receptor molecule can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducingt-PALP-induced activities, thereby preventing the action of t-PALP byexcluding t-PALP from binding.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed, for example, in Okano, J. Neurochem. 56: 560 (1991);“Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression.” CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooneyet al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360(1991). The methods are based on binding of a polynucleotide to acomplementary DNA or RNA. For example, the 5′ coding portion of apolynucleotide that encodes the mature polypeptide of the presentinvention may be used to design an antisense RNA oligonucleotide of fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcriptionthereby preventing transcription and the production of t-PALP. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into t-PALP polypeptide. Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of t-PALP protein.

The agonists and antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described above.

The antagonists may be employed for instance to inhibit t-PALPactivities such as fibrin binding. By inhibition of fibrin binding, at-PALP antagonist may decrease the efficacy of t-PALP enzymaticactivity. Such an inhibition may of interest if it is desirable tonegatively alter t-PALP activity in an indirect manner. Rather thandirectly targeting the active site of the t-PALP enzyme, it may be ofinterest to alter the activity of the enzyme by targeting itsfibrin-binding activity. Furthermore, t-PALP may be of use in regulatingthe proteolytic activity plasminogen. An antagonist which functions bydirectly binding to the t-PALP active site may reduce the localconcentration of functional plasminogen in a given system. Such acapability may desired as an effective means of ameliorating a currenttreatment procedure which has artificially increased the effectiveconcentration of plasminogen. In addition, the use of such a t-PALPantagonist may be used effectively to treat a system which has acongenitally increased level of t-PALP, and in turn, plasminogenactivity. Similarly, antibodies against t-PALP may be employed to bindto and inhibit t-PALP activity to treat the same or a related condition.Any of the above antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

Gene Mapping

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a t-PALP protein gene. Thiscan be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose.

In addition, in some cases, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15–25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Fluorescence in situ hybridization (“FISH”) of a cDNA cloneto a metaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance In Man, available on-line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Uses of the t-PALP Polynucleotides

t-PALP polynucleotides and/or polypeptides of the present invention, andagonists thereof, can be used as anti-angiogenesis, anti-tumorigenesis,and/or anti-cancer agents. As detailed in Examples 40, 52, and 53,t-PALP polynucleotides and polypeptides of the invention were used toinhibit the growth of TSU cells in two different tumor model systems.Despite the high tumorigenicity of TSU cells, treatment with t-PALP ofthe present invention resulted in a marked inhibition of tumor massesgrown on chick embryo chorioallantoic membranes (CAM); see Example 40.Additionally, t-PALP of the present invention also resulted in a markedinhibition on the growth rate of TSU cell xenograft tumors in athymicnude mice (Examples 51 and 52). t-PALP, and/or a mutein thereof, and/oran agonist, and/or an antagonist thereof, of the present invention, canbe used to treat a number of cancers including, but not limited to,breast cancer, colon cancer, cardiac tumors, pancreatic cancer,melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, adenoma, and thelike.

The t-PALP polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

There exists an ongoing need to identify new chromosome markers, sincefew chromosome marking reagents, based on actual sequence data (repeatpolymorphisms), are presently available.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15–25 bp) from the sequences shown in SEQ ID NO:1. Primerscan be selected using computer analysis so that primers do not span morethan one predicted exon in the genomic DNA. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human t-PALP genecorresponding to the SEQ ID NO:1 will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping thepolynucleotides to particular chromosomes. Three or more clones can beassigned per day using a single thermal cycler. Moreover,sublocalization of the t-PALP polynucleotides can be achieved withpanels of specific chromosome fragments. Other gene mapping strategiesthat can be used include in situ hybridization, prescreening withlabeled flow-sorted chromosomes, and preselection by hybridization toconstruct chromosome specific-cDNA libraries.

Precise chromosomal location of the t-PALP polynucleotides can also beachieved using fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000–4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the t-PALP polynucleotides can be usedindividually (to mark a single chromosome or a single site on thatchromosome) or in panels (for marking multiple sites and/or multiplechromosomes). Preferred polynucleotides correspond to the noncodingregions of the cDNAs because the coding sequences are more likelyconserved within gene families, thus increasing the chance of crosshybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location,the physical position of the polynucleotide can be used in linkageanalysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library).) Assuming 1 megabase mapping resolution and onegene per 20 kb, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50–500 potential causativegenes.

Thus, once coinheritance is established, differences in the t-PALPpolynucleotide and the corresponding gene between affected andunaffected individuals can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected individuals, but not in normalindividuals, indicates that the mutation may cause the disease. However,complete sequencing of the t-PALP polypeptide and the corresponding genefrom several normal individuals is required to distinguish the mutationfrom a polymorphism. If a new polymorphism is identified, thispolymorphic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affectedindividuals as compared to unaffected individuals can be assessed usingt-PALP polynucleotides. Any of these alterations (altered expression,chromosomal rearrangement, or mutation) can be used as a diagnostic orprognostic marker.

Thus, the invention also provides a diagnostic method useful duringdiagnosis of a disorder, involving measuring the expression level ofpolynucleotides of the present invention in cells or body fluid from anindividual and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

In still another embodiment, the invention includes a kit for analyzingsamples for the presence of proliferative and/or cancerouspolynucleotides derived from a test subject. In a general embodiment,the kit includes at least one polynucleotide probe containing anucleotide sequence that will specifically hybridize with apolynucleotide of the present invention and a suitable container. In aspecific embodiment, the kit includes two polynucleotide probes definingan internal region of the polynucleotide of the present invention, whereeach probe has one strand containing a 31′mer-end internal to theregion. In a further embodiment, the probes may be useful as primers forpolymerase chain reaction amplification.

Where a diagnosis of a disorder, has already been made according toconventional methods, the present invention is useful as a prognosticindicator, whereby patients exhibiting enhanced or depressedpolynucleotide of the present invention expression will experience aworse clinical outcome relative to patients expressing the gene at alevel nearer the standard level.

By “measuring the expression level of polynucleotide of the presentinvention” is intended qualitatively or quantitatively measuring orestimating the level of the polypeptide of the present invention or thelevel of the mRNA encoding the polypeptide in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of individuals nothaving a disorder. As will be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains the polypeptide of the present invention or mRNA. Asindicated, biological samples include body fluids (such as semen, lymph,sera, plasma, urine, synovial fluid and spinal fluid) which contain thepolypeptide of the present invention, and other tissue sources found toexpress the polypeptide of the present invention. Methods for obtainingtissue biopsies and body fluids from mammals are well known in the art.Where the biological sample is to include mRNA, a tissue biopsy is thepreferred source.

The method(s) provided above may preferrably be applied in a diagnosticmethod and/or kits in which polynucleotides and/or polypeptides areattached to a solid support. In one exemplary method, the support may bea “gene chip” or a “biological chip” as described in U.S. Pat. Nos.5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip withpolynucleotides of the present invention attached may be used toidentify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingcancerous diseases and conditions. Such a method is described in U.S.Pat. Nos. 5,858,659 and 5,856,104. The US Patents referenced supra arehereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the strong binding. In addition, it is morelikely that single base mismatches can be determined with PNA/DNAhybridization because a single mismatch in a PNA/DNA 15-mer lowers themelting point (T_(m)) by 8°–20° C., vs. 4°–16° C. for the DNA/DNA 15-merduplex. Also, the absence of charge groups in PNA means thathybridization can be done at low ionic strengths and reduce possibleinterference by salt during the analysis.

The present invention is useful for detecting cancer in mammals. Inparticular the invention is useful during diagnosis of pathological cellproliferative neoplasias which include, but are not limited to: acutemyelogenous leukemias including acute monocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute erythroleukemia, acute megakaryocyticleukemia, and acute undifferentiated leukemia, etc.; and chronicmyelogenous leukemias including chronic myelomonocytic leukemia, chronicgranulocytic leukemia, etc. Preferred mammals include monkeys, apes,cats, dogs, cows, pigs, horses, rabbits and humans. Particularlypreferred are humans.

Pathological cell proliferative disorders are often associated withinappropriate activation of proto-oncogenes. (Gelmann, E. P. et al.,“The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology,”in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds.,161–182 (1985)). Neoplasias are now believed to result from thequalitative alteration of a normal cellular gene product, or from thequantitative modification of gene expression by insertion into thechromosome of a viral sequence, by chromosomal translocation of a geneto a more actively transcribed region, or by some other mechanism.(Gelmann et al., supra) It is likely that mutated or altered expressionof specific genes is involved in the pathogenesis of some leukemias,among other tissues and cell types. (Gelmann et al., supra) Indeed, thehuman counterparts of the oncogenes involved in some animal neoplasiashave been amplified or translocated in some cases of human leukemia andcarcinoma. (Gelmann et al., supra)

For example, c-myc expression is highly amplified in the non-lymphocyticleukemia cell line HL-60. When HL-60 cells are chemically induced tostop proliferation, the level of c-myc is found to be downregulated.(International Publication Number WO 91/15580) However, it has beenshown that exposure of HL-60 cells to a DNA construct that iscomplementary to the 5′ end of c-myc or c-myb blocks translation of thecorresponding mRNAs which downregulates expression of the c-myc or c-mybproteins and causes arrest of cell proliferation and differentiation ofthe treated cells. (International Publication Number WO 91/15580;Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al.,Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisanwould appreciate the present invention's usefulness would not be limitedto treatment of proliferative disorders of hematopoietic cells andtissues, in light of the numerous cells and cell types of varyingorigins which are known to exhibit proliferative phenotypes.

In addition to the foregoing, a t-PALP polynucleotide can be used tocontrol gene expression through triple helix formation or antisense DNAor RNA. Antisense techniques are discussed, for example, in Okano, J.Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Triple helix formation is discussed in, for instance Lee et al., NucleicAcids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988);and Dervan et al., Science 251: 1360 (1991). Both methods rely onbinding of the polynucleotide to a complementary DNA or RNA. For thesetechniques, preferred polynucleotides are usually oligonucleotides 20 to40 bases in length and complementary to either the region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat disease.

t-PALP polynucleotides are also useful in gene therapy. One goal of genetherapy is to insert a normal gene into an organism having a defectivegene, in an effort to correct the genetic defect. t-PALP offers a meansof targeting such genetic defects in a highly accurate manner. Anothergoal is to insert a new gene that was not present in the host genome,thereby producing a new trait in the host cell.

The t-PALP polynucleotides are also useful for identifying individualsfrom minute biological samples. The United States military, for example,is considering the use of restriction fragment length polymorphism(RFLP) for identification of its personnel. In this technique, anindividual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentifying personnel. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The t-PALP polynucleotides can beused as additional DNA markers for RFLP.

The t-PALP polynucleotides can also be used as an alternative to RFLP,by determining the actual base-by-base DNA sequence of selected portionsof an individual's genome. These sequences can be used to prepare PCRprimers for amplifying and isolating such selected DNA, which can thenbe sequenced. Using this technique, individuals can be identifiedbecause each individual will have a unique set of DNA sequences. Once anunique ID database is established for an individual, positiveidentification of that individual, living or dead, can be made fromextremely small tissue samples.

Forensic biology also benefits from using DNA-based identificationtechniques as disclosed herein. DNA sequences taken from very smallbiological samples such as tissues, e.g., hair or skin, or body fluids,e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk,lymph, pulmonary sputum or surfactant, urine, fecal matter, etc., can beamplified using PCR. In one prior art technique, gene sequencesamplified from polymorphic loci, such as DQa class II HLA gene, are usedin forensic biology to identify individuals. (Erlich, H., PCRTechnology, Freeman and Co. (1992).) Once these specific polymorphicloci are amplified, they are digested with one or more restrictionenzymes, yielding an identifying set of bands on a Southern blot probedwith DNA corresponding to the DQa class II HLA gene. Similarly, t-PALPpolynucleotides can be used as polymorphic markers for forensicpurposes.

There is also a need for reagents capable of identifying the source of aparticular tissue. Such need arises, for example, in forensics whenpresented with tissue of unknown origin. Appropriate reagents cancomprise, for example, DNA probes or primers specific to particulartissue prepared from t-PALP sequences. Panels of such reagents canidentify tissue by species and/or by organ type. In a similar fashion,these reagents can be used to screen tissue cultures for contamination.

Because t-PALP is found expressed in cerebellum, smooth muscle, restingand PHA-treated T-cells, GM-CSF-treated macrophages, frontal cortex ofthe brain, breast lymph node, chronic lymphocytic leukemic spleen, andseveral other tissues, t-PALP polynucleotides are useful ashybridization probes for differential identification of the tissue(s) orcell type(s) present in a biological sample. Similarly, polypeptides andantibodies directed to t-PALP polypeptides are useful to provideimmunological probes for differential identification of the tissue(s) orcell type(s). In addition, for a number of disorders of the abovetissues or cells, particularly of the circulatory system, significantlyhigher or lower levels of t-PALP gene expression may be detected incertain tissues (e.g., cancerous and wounded tissues) or bodily fluids(e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken froman individual having such a disorder, relative to a “standard” t-PALPgene expression level, i.e., the t-PALP expression level in healthytissue from an individual not having the circulatory system disorder.

Thus, the invention provides a diagnostic method of a disorder, whichinvolves: (a) assaying t-PALP gene expression level in cells or bodyfluid of an individual; (b) comparing the t-PALP gene expression levelwith a standard t-PALP gene expression level, whereby an increase ordecrease in the assayed t-PALP gene expression level compared to thestandard expression level is indicative of a disorder in the circulatorysystem.

In the very least, the t-PALP polynucleotides can be used as molecularweight markers on Southern gels, as diagnostic probes for the presenceof a specific mRNA in a particular cell type, as a probe to“subtract-out” known sequences in the process of discovering novelpolynucleotides, for selecting and making oligomers for attachment to a“gene chip” or other support, to raise anti-DNA antibodies using DNAimmunization techniques, and as an antigen to elicit an immune response.

Uses of t-PALP Polypeptides

t-PALP polypeptides can be used in numerous ways. The followingdescription should be considered exemplary and utilizes knowntechniques.

t-PALP polypeptides can be used to assay protein levels in a biologicalsample using antibody-based techniques. For example, protein expressionin tissues can be studied with classical immunohistological methods.(Jalkanen, M., et al., J. Cell. Biol. 101:976–985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087–3096 (1987).) Other antibody-basedmethods useful for detecting protein gene expression includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA). Suitable antibody assay labels are known inthe art and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S),tritium (3H), indium (112In), and technetium (99 mTc), and fluorescentlabels, such as fluorescein and rhodamine, and biotin.

In addition to assaying protein levels in a biological sample, proteinscan also be detected in vivo by imaging. Antibody labels or markers forin vivo imaging of protein include those detectable by X-radiography,NMR or ESR. For X-radiography, suitable labels include radioisotopessuch as barium or cesium, which emit detectable radiation but are notovertly harmful to the subject. Suitable markers for NMR and ESR includethose with a detectable characteristic spin, such as deuterium, whichmay be incorporated into the antibody by labeling of nutrients for therelevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, 131I, 112In, 99mTc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99 mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, whichinvolves (a) assaying the expression of t-PALP polypeptide in cells orbody fluid of an individual; (b) comparing the level of gene expressionwith a standard gene expression level, whereby an increase or decreasein the assayed t-PALP polypeptide gene expression level compared to thestandard expression level is indicative of a disorder. With respect tocancer, the presence of a relatively high amount of transcript inbiopsied tissue from an individual may indicate a predisposition for thedevelopment of the disease, or may provide a means for detecting thedisease prior to the appearance of actual clinical symptoms. A moredefinitive diagnosis of this type may allow health professionals toemploy preventative measures or aggressive treatment earlier therebypreventing the development or further progression of the cancer.

Moreover, t-PALP polypeptides can be used to treat disease. For example,patients can be administered t-PALP polypeptides in an effort to replaceabsent or decreased levels of the t-PALP polypeptide (e.g., insulin), tosupplement absent or decreased levels of a different polypeptide (e.g.,hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), toinhibit the activity of a polypeptide (e.g., an oncogene or tumorsupressor), to activate the activity of a polypeptide (e.g., by bindingto a receptor), to reduce the activity of a membrane bound receptor bycompeting with it for free ligand (e.g., soluble TNF receptors used inreducing inflammation), or to bring about a desired response (e.g.,blood vessel growth inhibition, enhancement of the immune response toproliferative cells or tissues).

Similarly, antibodies directed to t-PALP polypeptides can also be usedto treat disease. For example, administration of an antibody directed toa t-PALP polypeptide can bind and reduce overproduction of thepolypeptide. Similarly, administration of an antibody can activate thepolypeptide, such as by binding to a polypeptide bound to a membrane(receptor).

At the very least, the t-PALP polypeptides can be used as molecularweight markers on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art. t-PALPpolypeptides can also be used to raise antibodies, which in turn areused to measure protein expression from a recombinant cell, as a way ofassessing transformation of the host cell. Moreover, t-PALP polypeptidescan be used to test the following biological activities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating disorders, diseases and conditions. The gene therapy methodsrelate to the introduction of nucleic acid (DNA, RNA and antisense DNAor RNA) sequences into an animal to achieve expression of the t-PALPpolypeptide of the present invention. This method requires apolynucleotide which codes for a t-PALP polypeptide operatively linkedto a promoter and any other genetic elements necessary for theexpression of the polypeptide by the target tissue. Such gene therapyand delivery techniques are known in the art, see, for example,WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to at-PALP polynucleotide ex vivo, with the engineered cells then beingprovided to a patient to be treated with the polypeptide. Such methodsare well-known in the art. For example, see Belldegrun, A., et al., J.Natl. Cancer Inst. 85: 207–216 (1993); Ferrantini, M. et al., CancerResearch 53: 1107–1112 (1993); Ferrantini, M. et al., J. Immunology 153:4604–4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221–229 (1995);Ogura, H., et al., Cancer Research 50: 5102–5106 (1990); Santodonato,L., et al., Human Gene Therapy 7:1–10 (1996); Santodonato, L., et al.,Gene Therapy 4:1246–1255 (1997); and Zhang, J.-F. et al., Cancer GeneTherapy 3: 31–38 (1996)), which are herein incorporated by reference. Inone embodiment, the cells which are engineered are arterial cells. Thearterial cells may be reintroduced into the patient through directinjection to the artery, the tissues surrounding the artery, or throughcatheter injection.

As discussed in more detail below, the t-PALP polynucleotide constructscan be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, and the like). The t-PALPpolynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

In one embodiment, the t-PALP polynucleotide is delivered as a nakedpolynucleotide. The term “naked” polynucleotide, DNA or RNA refers tosequences that are free from any delivery vehicle that acts to assist,promote or facilitate entry into the cell, including viral sequences,viral particles, liposome formulations, lipofectin or precipitatingagents and the like. However, the t-PALP polynucleotides can also bedelivered in liposome formulations and lipofectin formulations and thelike can be prepared by methods well known to those skilled in the art.Such methods are described, for example, in U.S. Pat. Nos. 5,593,972,5,589,466, and 5,580,859, which are herein incorporated by reference.

The t-PALP polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication.Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; pSVK3, pBPV, pMSG and pSVL available fromPharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of t-PALP polynucleotide sequence. Suitablepromoters include adenoviral promoters, such as the adenoviral majorlate promoter; or heterologous promoters, such as the cytomegalovirus(CMV) promoter; the respiratory syncytial virus (RSV) promoter;inducible promoters, such as the MMT promoter, the metallothioneinpromoter; heat shock promoters; the albumin promoter; the ApoAIpromoter; human globin promoters; viral thymidine kinase promoters, suchas the Herpes Simplex thymidine kinase promoter; retroviral LTRs; theb-actin promoter; and human growth hormone promoters. The promoter alsomay be the native promoter for t-PALP.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The t-PALP polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 mg/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked t-PALP DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the t-PALP polynucleotide constructs arecomplexed in a liposome preparation. Liposomal preparations for use inthe instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA(1987) 84:7413–7416, which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077–6081, which isherein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem. (1990) 265:10189–10192, which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl. Acad. Sci. USA (1987) 84:7413–7416, which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413–7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512–527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA (1979) 76:145); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75:145;Schaefer-Ridder et al., Science (1982) 215:166), which are hereinincorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication no. WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, usinga retroviral particle containing RNA which comprises a sequence encodingt-PALP. Retroviruses from which the retroviral plasmid vectors may bederived include, but are not limited to, Moloney Murine Leukemia Virus,spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avianleukosis virus, gibbon ape leukemia virus, human immunodeficiency virus,Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14×, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5–14 (1990),which is incorporated herein by reference in its entirety. The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding t-PALP. Such retroviral vectorparticles then may be employed, to transduce eukaryotic cells, either invitro or in vivo. The transduced eukaryotic cells will express t-PALP.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with t-PALP polynucleotide contained in an adenovirus vector. Adenoviruscan be manipulated such that it encodes and expresses t-PALP, and at thesame time is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. Adenovirus expression is achieved withoutintegration of the viral DNA into the host cell chromosome, therebyalleviating concerns about insertional mutagenesis. Furthermore,adenoviruses have been used as live enteric vaccines for many years withan excellent safety profile (Schwartz, A. R. et al. (1974) Am. Rev.Respir. Dis.109:233–238). Finally, adenovirus mediated gene transfer hasbeen demonstrated in a number of instances including transfer ofalpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al. (1991) Science 252:431–434; Rosenfeld et al., (1992) Cell68:143–155). Furthermore, extensive studies to attempt to establishadenovirus as a causative agent in human cancer were uniformly negative(Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499–503 (1993); Rosenfeld et al., Cell 68:143–155 (1992);Engelhardt et al., Human Genet. Ther. 4:759–769 (1993); Yang et al.,Nature Genet. 7:362–369 (1994); Wilson et al., Nature 365:691–692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Imunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The t-PALP polynucleotideconstruct is inserted into the AAV vector using standard cloningmethods, such as those found in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAVvector is then transfected into packaging cells which are infected witha helper virus, using any standard technique, including lipofection,electroporation, calcium phosphate precipitation, etc. Appropriatehelper viruses include adenoviruses, cytomegaloviruses, vacciniaviruses, or herpes viruses. Once the packaging cells are transfected andinfected, they will produce infectious AAV viral particles which containthe t-PALP polynucleotide construct. These viral particles are then usedto transduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the t-PALP polynucleotide construct integrated intoits genome, and will express t-PALP.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding t-PALP) via homologous recombination (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932–8935 (1989); and Zijlstra et al., Nature 342:435–438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not normally expressed in the cells, or isexpressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the t-PALP desired endogenous polynucleotide sequenceso the promoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous t-PALP sequence is placedunder the control of the promoter. The promoter then drives theexpression of the endogenous t-PALP sequence.

The polynucleotides encoding t-PALP may be administered along with otherpolynucleotides encoding an angiogenic protein. Examples of angiogenicproteins include, but are not limited to, acidic and basic fibroblastgrowth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alphaand beta, platelet-derived endothelial cell growth factor,platelet-derived growth factor, tumor necrosis factor alpha, hepatocytegrowth factor, insulin like growth factor, colony stimulating factor,macrophage colony stimulating factor, granulocyte/macrophage colonystimulating factor, and nitric oxide synthase.

Preferably, the polynucleotide encoding t-PALP contains a secretorysignal sequence that facilitates secretion of the protein. Typically,the signal sequence is positioned in the coding region of thepolynucleotide to be expressed towards or at the 5′ end of the codingregion. The signal sequence may be homologous or heterologous to thepolynucleotide of interest and may be homologous or heterologous to thecells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers (Kaneda et al.,Science 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277–11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian.

Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly preferred.

Biological Activities of t-PALP

t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, can be used in assays to test for one or more biologicalactivities. If t-PALP polynucleotides or polypeptides, or agonists orantagonists of t-PALP, do exhibit activity in a particular assay, it islikely that t-PALP may be involved in the diseases associated with thebiological activity. Therefore, t-PALP could be used to treat theassociated disease.

Immune Activity

t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, may be useful in treating deficiencies or disorders of theimmune system, by activating or inhibiting the proliferation,differentiation, or mobilization (chemotaxis) of immune cells. Immunecells develop through a process called hematopoiesis, producing myeloid(platelets, red blood cells, neutrophils, and macrophages) and lymphoid(B and T lymphocytes) cells from pluripotent stem cells. The etiology ofthese immune deficiencies or disorders may be genetic, somatic, such ascancer or some autoimmune disorders, acquired (e.g., by chemotherapy ortoxins), or infectious. Moreover, t-PALP polynucleotides orpolypeptides, or agonists or antagonists of t-PALP, can be used as amarker or detector of a particular immune system disease or disorder.

t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, may be useful in treating or detecting deficiencies or disordersof hematopoietic cells. t-PALP polynucleotides or polypeptides, oragonists or antagonists of t-PALP, could be used to increasedifferentiation and proliferation of hematopoietic cells, including thepluripotent stem cells, in an effort to treat those disorders associatedwith a decrease in certain (or many) types hematopoietic cells. Examplesof immunologic deficiency syndromes include, but are not limited to:blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia),ataxia telangiectasia, common variable immunodeficiency, DigeorgeSyndrome, HIV infection, HTLV-BLV infection, leukocyte adhesiondeficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction,severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder,anemia, thrombocytopenia, or hemoglobinuria.

Moreover, t-PALP polynucleotides or polypeptides, or agonists orantagonists of t-PALP, can also be used to modulate hemostatic (thestopping of bleeding) or thrombolytic activity (clot formation). Forexample, by increasing hemostatic or thrombolytic activity, t-PALPpolynucleotides or polypeptides, or agonists or antagonists of t-PALP,could be used to treat blood coagulation disorders (e.g.,afibrinogenemia, factor deficiencies), blood platelet disorders (e.g.thrombocytopenia), or wounds resulting from trauma, surgery, or othercauses. Alternatively, t-PALP polynucleotides or polypeptides, oragonists or antagonists of t-PALP, that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting.These molecules could be important in the treatment of heart attacks(infarction), strokes, or scarring.

t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, may also be useful in treating or detecting autoimmunedisorders. Many autoimmune disorders result from inappropriaterecognition of self as foreign material by immune cells. Thisinappropriate recognition results in an immune response leading to thedestruction of the host tissue. Therefore, the administration of t-PALPpolynucleotides or polypeptides, or agonists or antagonists of t-PALP,that can inhibit an immune response, particularly the proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing autoimmune disorders.

Examples of autoimmune disorders that can be treated or detectedinclude, but are not limited to: Addison's Disease, hemolytic anemia,antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergicencephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves'Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia,Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter'sDisease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic LupusErythematosus, Autoimmune Pulmonary Inflammation, Guillain-BarreSyndrome, insulin dependent diabetes mellitis, and autoimmuneinflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by t-PALP polynucleotides or polypeptides, or agonists orantagonists of t-PALP. Moreover, these molecules can be used to treatanaphylaxis, hypersensitivity to an antigenic molecule, or blood groupincompatibility.

t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, may also be used to treat and/or prevent organ rejection orgraft-versus-host disease (GVHD). Organ rejection occurs by host immunecell destruction of the transplanted tissue through an immune response.Similarly, an immune response is also involved in GVHD, but, in thiscase, the foreign transplanted immune cells destroy the host tissues.The administration of t-PALP polynucleotides or polypeptides, oragonists or antagonists of t-PALP, that inhibits an immune response,particularly the proliferation, differentiation, or chemotaxis ofT-cells, may be an effective therapy in preventing organ rejection orGVHD.

Similarly, t-PALP polynucleotides or polypeptides, or agonists orantagonists of t-PALP, may also be used to modulate inflammation. Forexample, t-PALP polynucleotides or polypeptides, or agonists orantagonists of t-PALP, may inhibit the proliferation and differentiationof cells involved in an inflammatory response. These molecules can beused to treat inflammatory conditions, both chronic and acuteconditions, including chronic prostatitis, granulomatous prostatitis andmalacoplakia, inflammation associated with infection (e.g., septicshock, sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

Hyperproliferative Disorders

t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, can be used to treat or detect hyperproliferative disorders,including neoplasms. t-PALP polynucleotides or polypeptides, or agonistsor antagonists of t-PALP, may inhibit the proliferation of the disorderthrough direct or indirect interactions. Alternatively, t-PALPpolynucleotides or polypeptides, or agonists or antagonists of t-PALP,may proliferate other cells which can inhibit the hyperproliferativedisorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

Examples of hyperproliferative disorders that can be treated or detectedby t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, include, but are not limited to neoplasms located in the: colon,abdomen, bone, breast, digestive system, liver, pancreas, peritoneum,endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary,thymus, thyroid), eye, head and neck, nervous (central and peripheral),lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, andurogenital.

Similarly, other hyperproliferative disorders can also be treated ordetected by t-PALP polynucleotides or polypeptides, or agonists orantagonists of t-PALP. Examples of such hyperproliferative disordersinclude, but are not limited to:

hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,Gaucher's Disease, histiocytosis, and any other hyperproliferativedisease, besides neoplasia, located in an organ system listed above.

One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating cellproliferative disorders by inserting into an abnormally proliferatingcell a polynucleotide of the present invention, wherein saidpolynucleotide represses said expression.

Another embodiment of the present invention provides a method oftreating cell-proliferative disorders in individuals comprisingadministration of one or more active gene copies of the presentinvention to an abnormally proliferating cell or cells. In a preferredembodiment, polynucleotides of the present invention is a DNA constructcomprising a recombinant expression vector effective in expressing a DNAsequence encoding said polynucleotides. In another preferred embodimentof the present invention, the DNA construct encoding the polynucleotidesof the present invention is inserted into cells to be treated utilizinga retrovirus, or more preferrably an adenoviral vector (See G J. Nabel,et. al., PNAS 1999 96: 324–326, which is hereby incorporated byreference). In a most preferred embodiment, the viral vector isdefective and will not transform non-proliferating cells, onlyproliferating cells. Moreover, in a preferred embodiment, thepolynucleotides of the present invention inserted into proliferatingcells either alone, or in combination with or fused to otherpolynucleotides, can then be modulated via an external stimulus (i.e.magnetic, specific small molecule, chemical, or drug administration,etc.), which acts upon the promoter upstream of said polynucleotides toinduce expression of the encoded protein product. As such the beneficialtherapeutic affect of the present invention may be expressly modulated(i.e. to increase, decrease, or inhibit expression of the presentinvention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressingexpression of oncogenic genes or antigens. By “repressing expression ofthe oncogenic genes” is intended the suppression of the transcription ofthe gene, the degradation of the gene transcript (pre-message RNA), theinhibition of splicing, the destruction of the messenger RNA, theprevention of the post-translational modifications of the protein, thedestruction of the protein, or the inhibition of the normal function ofthe protein.

For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directlyto cell proliferative disorder/disease sites in internal organs, bodycavities and the like by use of imaging devices used to guide aninjecting needle directly to the disease site. The polynucleotides ofthe present invention may also be administered to disease sites at thetime of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease ordisorder, affecting any one or any combination of organs, cavities, orbody parts, which is characterized by single or multiple local abnormalproliferations of cells, groups of cells, or tissues, whether benign ormalignant.

Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapieswhich involve administering of anti-polypeptides and anti-polynucleotideantibodies to a mammalian, preferably human, patient for treating one ormore of the described disorders. Methods for producing anti-polypeptidesand anti-polynucleotide antibodies polyclonal and monoclonal antibodiesare described in detail elsewhere herein. Such antibodies may beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the presentinvention are useful for treating a subject having or developing cellproliferative and/or differentiation disorders as described herein. Suchtreatment comprises administering a single or multiple doses of theantibody, or a fragment, derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example, which serve toincrease the number or activity of effector cells which interact withthe antibodies.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragements thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragements thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M,10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M,10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M,and 10⁻¹⁵M.

Moreover, polypeptides of the present invention are useful in inhibitingthe angiogenesis of proliferative cells or tissues, either alone, as aprotein fusion, or in combination with other polypeptides directly orindirectly, as described elsewhere herein. In a most preferredembodiment, said anti-angiogenesis effect may be achieved indirectly,for example, through the inhibition of hematopoietic, tumor-specificcells, such as tumor-associated macrophages (See Joseph I B, et al. JNatl Cancer Inst, 90(21):1648–53 (1998), which is hereby incorporated byreference). Antibodies directed to polypeptides or polynucleotides ofthe present invention may also result in inhibition of angiogenesisdirectly, or indirectly (See Witte L, et al., Cancer Metastasis Rev.17(2):155–61 (1998), which is hereby incorporated by reference)).

Polypeptides, including protein fusions, of the present invention, orfragments thereof may be useful in inhibiting proliferative cells ortissues through the induction of apoptosis. Said polypeptides may acteither directly, or indirectly to induce apoptosis of proliferativecells and tissues, for example in the activation of a death-domainreceptor, such as tumor necrosis factor (TNF) receptor-1, CD95(Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) andTNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (SeeSchulze-Osthoff K, et. al., Eur J Biochem 254(3):439–59 (1998), which ishereby incorporated by reference). Moreover, in another preferredembodiment of the present invention, said polypeptides may induceapoptosis through other mechanisms, such as in the activation of otherproteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuviants, such as apoptonin, galectins,thioredoxins, antiinflammatory proteins (See for example, Mutat Res400(1–2):447–55 (1998), Med Hypotheses.50(5):423–33 (1998), Chem BiolInteract. April 24;111–112:23–34 (1998), J Mol Med.76(6):402–12 (1998),Int J Tissue React;20(1):3–15 (1998), which are all hereby incorporatedby reference).

Polypeptides, including protein fusions to, or fragments thereof, of thepresent invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol hnmunol1998;231:125–41, which is hereby incorporated by reference). Suchthereapeutic affects of the present invention may be achieved eitheralone, or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing polypeptides or polypeptide antibodes associatedwith heterologous polypeptides, heterologous nucleic acids, toxins, orprodrugs) to targeted cells expressing the polypeptide of the presentinvention. Polypeptides or polypeptide antibodes of the invention may beassociated with with heterologous polypeptides, heterologous nucleicacids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/orcovalent interactions.

Polypeptides, protein fusions to, or fragments thereof, of the presentinvention are useful in enhancing the immunogenicity and/or antigenicityof proliferating cells or tissues, either directly, such as would occurif the polypeptides of the present invention ‘vaccinated’ the immuneresponse to respond to proliferative antigens and immunogens, orindirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

Cardiovascular Disorders

t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, encoding t-PALP may be used to treat cardiovascular disorders,including peripheral artery disease, such as limb ischemia.

Cardiovascular disorders include cardiovascular abnormalities, such asarterio-arterial fistula, arteriovenous fistula, cerebral arteriovenousmalformations, congenital heart defects, pulmonary atresia, and ScimitarSyndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia,patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex,hypoplastic left heart syndrome, levocardia, tetralogy of fallot,transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, suchas aortopulmonary septal defect, endocardial cushion defects,Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septaldefects.

Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include carotid artery diseases, cerebralamyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebralarteriosclerosis, cerebral arteriovenous malformation, cerebral arterydiseases, cerebral embolism and thrombosis, carotid artery thrombosis,sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epiduralhematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebralinfarction, cerebral ischemia (including transient), subclavian stealsyndrome, periventricular leukomalacia, vascular headache, clusterheadache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, are especially effective for the treatment of critical limbischemia and coronary disease.

t-PALP polypeptides may be administered using any method known in theart, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, biolistic injectors, particle accelerators, gelfoam spongedepots, other commercially available depot materials, osmotic pumps,oral or suppositorial solid pharmaceutical formulations, decanting ortopical applications during surgery, aerosol delivery. Such methods areknown in the art. t-PALP polypeptides may be administered as part of aTherapeutic, described in more detail below. Methods of deliveringt-PALP polynucleotides are described in more detail herein.

Anti-Angiogenesis Activity

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345–355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630–634 (1991); Folkmanet al., N. Engl. J. Med., 333: 1757–1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401–411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175–203 (1985);Patz, Am. J. Opthalmol. 94:715–743 (1982); and Folkman et al., Science221:719–725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442–447 (1987).

The present invention provides for treatment of diseases or disordersassociated with neovascularization by administration of thepolynucleotides and/or polypeptides of the invention, as well asagonists or antagonists of the present invention. Malignant andmetastatic conditions which can be treated with the polynucleotides andpolypeptides, or agonists or antagonists of the invention include, butare not limited to, malignancies, solid tumors, and cancers describedherein and otherwise known in the art (for a review of such disorders,see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia(1985)). Thus, the present invention provides a method of treating anangiogenesis-related disease and/or disorder, comprising administeringto an individual in need thereof a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist of the invention.For example, polynucleotides, polypeptides, antagonists and/or agonistsmay be utilized in a variety of additional methods in order totherapeutically treat a cancer or tumor. Cancers which may be treatedwith polynucleotides, polypeptides, antagonists and/or agonists include,but are not limited to solid tumors, including prostate, lung, breast,ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid,biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,bladder, thyroid cancer; primary tumors and metastases; melanomas;glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lungcancer; colorectal cancer; advanced malignancies; and blood born tumorssuch as leukemias. For example, polynucleotides, polypeptides,antagonists and/or agonists may be delivered topically, in order totreat cancers such as skin cancer, head and neck tumors, breast tumors,and Kaposi's sarcoma.

Within yet other aspects, polynucleotides, polypeptides, antagonistsand/or agonists may be utilized to treat superficial forms of bladdercancer by, for example, intravesical administration. Polynucleotides,polypeptides, antagonists and/or agonists may be delivered directly intothe tumor, or near the tumor site, via injection or a catheter. Ofcourse, as the artisan of ordinary skill will appreciate, theappropriate mode of administration will vary according to the cancer tobe treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and/or agonists may be usefulin treating other disorders, besides cancers, which involveangiogenesis. These disorders include, but are not limited to: benigntumors, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; artheroscleric plaques; ocularangiogenic diseases, for example, diabetic retinopathy, retinopathy ofprematurity, macular degeneration, corneal graft rejection, neovascularglaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis andPterygia (abnormal blood vessel growth) of the eye; rheumatoidarthritis; psoriasis; delayed wound healing; endometriosis;vasculogenesis; granulations; hypertrophic scars (keloids); nonunionfractures; scleroderma; trachoma; vascular adhesions; myocardialangiogenesis; coronary collaterals; cerebral collaterals; arteriovenousmalformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;fibromuscular dysplasia; wound granulation; Crohn's disease; andatherosclerosis.

For example, within one aspect of the present invention methods areprovided for treating hypertrophic scars and keloids, comprising thestep of administering a polynucleotide, polypeptide, antagonist and/oragonist of the invention to a hypertrophic scar or keloid.

Within one embodiment of the present invention polynucleotides,polypeptides, antagonists and/or agonists are directly injected into ahypertrophic scar or keloid, in order to prevent the progression ofthese lesions. This therapy is of particular value in the prophylactictreatment of conditions which are known to result in the development ofhypertrophic scars and keloids (e.g., burns), and is preferablyinitiated after the proliferative phase has had time to progress(approximately 14 days after the initial injury), but beforehypertrophic scar or keloid development. As noted above, the presentinvention also provides methods for treating neovascular diseases of theeye, including for example, corneal neovascularization, neovascularglaucoma, proliferative diabetic retinopathy, retrolental fibroplasiaand macular degeneration.

Moreover, Ocular disorders associated with neovascularization which canbe treated with the polynucleotides and polypeptides of the presentinvention (including agonists and/or antagonists) include, but are notlimited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma,retrolental fibroplasia, uveitis, retinopathy of prematurity maculardegeneration, corneal graft neovascularization, as well as other eyeinflammatory diseases, ocular tumors and diseases associated withchoroidal or iris neovascularization. See, e.g., reviews by Waltman etal., Am. J. Ophthal. 85:704–710 (1978) and Gartner et al., Surv.Ophthal. 22:291–312 (1978).

Thus, within one aspect of the present invention methods are providedfor treating neovascular diseases of the eye such as cornealneovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of a compound (as described above) to the cornea, suchthat the formation of blood vessels is inhibited. Briefly, the cornea isa tissue which normally lacks blood vessels. In certain pathologicalconditions however, capillaries may extend into the cornea from thepericorneal vascular plexus of the limbus. When the cornea becomesvascularized, it also becomes clouded, resulting in a decline in thepatient's visual acuity. Visual loss may become complete if the corneacompletely opacitates. A wide variety of disorders can result in cornealneovascularization, including for example, corneal infections (e.g.,trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis),immunological processes (e.g., graft rejection and Stevens-Johnson'ssyndrome), alkali burns, trauma, inflammation (of any cause), toxic andnutritional deficiency states, and as a complication of wearing contactlenses.

Within particularly preferred embodiments of the invention, may beprepared for topical administration in saline (combined with any of thepreservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, anti-angiogenic compositions, prepared asdescribed above, may also be administered directly to the cornea. Withinpreferred embodiments, the anti-angiogenic composition is prepared witha muco-adhesive polymer which binds to cornea. Within furtherembodiments, the anti-angiogenic factors or anti-angiogenic compositionsmay be utilized as an adjunct to conventional steroid therapy. Topicaltherapy may also be useful prophylactically in corneal lesions which areknown to have a high probability of inducing an angiogenic response(such as chemical burns). In these instances the treatment, likely incombination with steroids, may be instituted immediately to help preventsubsequent complications.

Within other embodiments, the compounds described above may be injecteddirectly into the corneal stroma by an ophthalmologist under microscopicguidance. The preferred site of injection may vary with the morphologyof the individual lesion, but the goal of the administration would be toplace the composition at the advancing front of the vasculature (i.e.,interspersed between the blood vessels and the normal cornea). In mostcases this would involve perilimbic corneal injection to “protect” thecornea from the advancing blood vessels. This method may also beutilized shortly after a corneal insult in order to prophylacticallyprevent corneal neovascularization. In this situation the material couldbe injected in the perilimbic cornea interspersed between the corneallesion and its undesired potential limbic blood supply. Such methods mayalso be utilized in a similar fashion to prevent capillary invasion oftransplanted corneas. In a sustained-release form injections might onlybe required 2–3 times per year. A steroid could also be added to theinjection solution to reduce inflammation resulting from the injectionitself.

Within another aspect of the present invention, methods are provided fortreating neovascular glaucoma, comprising the step of administering to apatient a therapeutically effective amount of a polynucleotide,polypeptide, antagonist and/or agonist to the eye, such that theformation of blood vessels is inhibited. In one embodiment, the compoundmay be administered topically to the eye in order to treat early formsof neovascular glaucoma. Within other embodiments, the compound may beimplanted by injection into the region of the anterior chamber angle.Within other embodiments, the compound may also be placed in anylocation such that the compound is continuously released into theaqueous humor. Within another aspect of the present invention, methodsare provided for treating proliferative diabetic retinopathy, comprisingthe step of administering to a patient a therapeutically effectiveamount of a polynucleotide, polypeptide, antagonist and/or agonist tothe eyes, such that the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection into theaqueous humor or the vitreous, in order to increase the localconcentration of the polynucleotide, polypeptide, antagonist and/oragonist in the retina. Preferably, this treatment should be initiatedprior to the acquisition of severe disease requiring photocoagulation.

Within another aspect of the present invention, methods are provided fortreating retrolental fibroplasia, comprising the step of administeringto a patient a therapeutically effective amount of a polynucleotide,polypeptide, antagonist and/or agonist to the eye, such that theformation of blood vessels is inhibited. The compound may beadministered topically, via intravitreous injection and/or viaintraocular implants.

Additionally, disorders which can be treated with the polynucleotides,polypeptides, agonists and/or agonists include, but are not limited to,hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,delayed wound healing, granulations, hemophilic joints, hypertrophicscars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

Moreover, disorders and/or states, which can be treated with be treatedwith the the polynucleotides, polypeptides, agonists and/or agonistsinclude, but are not limited to, solid tumors, blood born tumors such asleukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, forexample hemangiomas, acoustic neuromas, neurofibromas, trachomas, andpyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenicdiseases, for example, diabetic retinopathy, retinopathy of prematurity,macular degeneration, corneal graft rejection, neovascular glaucoma,retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayedwound healing, endometriosis, vascluogenesis, granulations, hypertrophicscars (keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compoundsufficient to block embryo implantation is administered before or afterintercourse and fertilization have occurred, thus providing an effectivemethod of birth control, possibly a “morning after” method.Polynucleotides, polypeptides, agonists and/or agonists may also be usedin controlling menstruation or administered as either a peritoneallavage fluid or for peritoneal implantation in the treatment ofendometriosis.

Polynucleotides, polypeptides, agonists and/or agonists of the presentinvention may be incorporated into surgical sutures in order to preventstitch granulomas.

Polynucleotides, polypeptides, agonists and/or agonists may be utilizedin a wide variety of surgical procedures. For example, within one aspectof the present invention a compositions (in the form of, for example, aspray or film) may be utilized to coat or spray an area prior to removalof a tumor, in order to isolate normal surrounding tissues frommalignant tissue, and/or to prevent the spread of disease to surroundingtissues. Within other aspects of the present invention, compositions(e.g., in the form of a spray) may be delivered via endoscopicprocedures in order to coat tumors, or inhibit angiogenesis in a desiredlocale. Within yet other aspects of the present invention, surgicalmeshes which have been coated with anti-angiogenic compositions of thepresent invention may be utilized in any procedure wherein a surgicalmesh might be utilized. For example, within one embodiment of theinvention a surgical mesh laden with an anti-angiogenic composition maybe utilized during abdominal cancer resection surgery (e.g., subsequentto colon resection) in order to provide support to the structure, and torelease an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are providedfor treating tumor excision sites, comprising administering apolynucleotide, polypeptide, agonist and/or agonist to the resectionmargins of a tumor subsequent to excision, such that the localrecurrence of cancer and the formation of new blood vessels at the siteis inhibited. Within one embodiment of the invention, theanti-angiogenic compound is administered directly to the tumor excisionsite (e.g., applied by swabbing, brushing or otherwise coating theresection margins of the tumor with the anti-angiogenic compound).Alternatively, the anti-angiogenic compounds may be incorporated intoknown surgical pastes prior to administration. Within particularlypreferred embodiments of the invention, the anti-angiogenic compoundsare applied after hepatic resections for malignancy, and afterneurosurgical operations.

Within one aspect of the present invention, polynucleotides,polypeptides, agonists and/or agonists may be administered to theresection margin of a wide variety of tumors, including for example,breast, colon, brain and hepatic tumors. For example, within oneembodiment of the invention, anti-angiogenic compounds may beadministered to the site of a neurological tumor subsequent to excision,such that the formation of new blood vessels at the site are inhibited.

The polynucleotides, polypeptides, agonists and/or agonists of thepresent invention may also be administered along with otheranti-angiogenic factors. Representative examples of otheranti-angiogenic factors include: Anti-Invasive Factor, retinoic acid andderivatives thereof, paclitaxel, Suramin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2,and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22–26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321–17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475–480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555–557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440–1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659–1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312–316, 1992); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole; andmetalloproteinase inhibitors such as BB94.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition ofapoptosis that could be treated or detected by t-PALP polynucleotides orpolypeptides, as well as antagonists or agonists of t-PALP, includecancers (such as follicular lymphomas, carcinomas with p53 mutations,and hormone-dependent tumors, including, but not limited to coloncancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomachcancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer);autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome,Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn'sdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection. Inpreferred embodiments, t-PALP polynucleotides, polypeptides, and/orantagonists of the invention are used to inhibit growth, progression,and/or metasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by t-PALP polynucleotides orpolypeptides, or agonists or antagonists of t-PALP, include, but are notlimited to, progression, and/or metastases of malignancies and relateddisorders such as leukemia (including acute leukemias (e.g., acutelymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis that could be treated ordetected by t-PALP polynucleotides or polypeptides, as well as agonistsor antagonists of t-PALP, include AIDS; neurodegenerative disorders(such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateralsclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumoror prior associated disease); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) myelodysplastic syndromes (such as aplastic anemia), graft v.host disease, ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), liver injury (e.g.,hepatitis related liver injury, ischemia/reperfusion injury, cholestosis(bile duct injury) and liver cancer); toxin-induced liver disease (suchas that caused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing t-PALP polynucleotides orpolypeptides, as well as agonists or antagonists of t-PALP, fortherapeutic purposes, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of dermal wounds.t-PALP polynucleotides or polypeptides, as well as agonists orantagonists of t-PALP, may be clinically useful in stimulating woundhealing including surgical wounds, excisional wounds, deep woundsinvolving damage of the dermis and epidermis, eye tissue wounds, dentaltissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resultingfrom heat exposure or chemicals, and other abnormal wound healingconditions such as uremia, malnutrition, vitamin deficiencies andcomplications associted with systemic treatment with steroids, radiationtherapy and antineoplastic drugs and antimetabolites. t-PALPpolynucleotides or polypeptides, as well as agonists or antagonists oft-PALP, could be used to promote dermal reestablishment subsequent todermal loss

t-PALP polynucleotides or polypeptides, as well as agonists orantagonists of t-PALP, could be used to increase the adherence of skingrafts to a wound bed and to stimulate re-epithelialization from thewound bed. The following are types of grafts that t-PALP polynucleotidesor polypeptides, agonists or antagonists of t-PALP, could be used toincrease adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepdermic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft, heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, thick split graft. t-PALPpolynucleotides or polypeptides, as well as agonists or antagonists oft-PALP, can be used to promote skin strength and to improve theappearance of aged skin.

It is believed that t-PALP polynucleotides or polypeptides, as well asagonists or antagonists of t-PALP, will also produce changes inhepatocyte proliferation, and epithelial cell proliferation in the lung,breast, pancreas, stomach, small intesting, and large intestine. t-PALPpolynucleotides or polypeptides, as well as agonists or antagonists oft-PALP, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. t-PALP polynucleotides or polypeptides, agonists or antagonistsof t-PALP, may promote proliferation of endothelial cells,keratinocytes, and basal keratinocytes.

t-PALP polynucleotides or polypeptides, as well as agonists orantagonists of t-PALP, could also be used to reduce the side effects ofgut toxicity that result from radiation, chemotherapy treatments orviral infections. t-PALP polynucleotides or polypeptides, as well asagonists or antagonists of t-PALP, may have a cytoprotective effect onthe small intestine mucosa. t-PALP polynucleotides or polypeptides, aswell as agonists or antagonists of t-PALP, may also stimulate healing ofmucositis (mouth ulcers) that result from chemotherapy and viralinfections.

t-PALP polynucleotides or polypeptides, as well as agonists orantagonists of t-PALP, could further be used in full regeneration ofskin in full and partial thickness skin defects, including burns, (i.e.,repopulation of hair follicles, sweat glands, and sebaceous glands),treatment of other skin defects such as psoriasis. t-PALPpolynucleotides or polypeptides, as well as agonists or antagonists oft-PALP, could be used to treat epidermolysis bullosa, a defect inadherence of the epidermis to the underlying dermis which results infrequent, open and painful blisters by accelerating reepithelializationof these lesions. t-PALP polynucleotides or polypeptides, as well asagonists or antagonists of t-PALP, could also be used to treat gastricand doudenal ulcers and help heal by scar formation of the mucosallining and regeneration of glandular mucosa and duodenal mucosal liningmore rapidly. Inflamamatory bowel diseases, such as Crohn's disease andulcerative colitis, are diseases which result in destruction of themucosal surface of the small or large intestine, respectively. Thus,t-PALP polynucleotides or polypeptides, as well as agonists orantagonists of t-PALP, could be used to promote the resurfacing of themucosal surface to aid more rapid healing and to prevent progression ofinflammatory bowel disease. Treatment with t-PALP polynucleotides orpolypeptides, agonists or antagonists of t-PALP, is expected to have asignificant effect on the production of mucus throughout thegastrointestinal tract and could be used to protect the intestinalmucosa from injurious substances that are ingested or following surgery.t-PALP polynucleotides or polypeptides, as well as agonists orantagonists of t-PALP, could be used to treat diseases associate withthe under expression of t-PALP.

Moreover, t-PALP polynucleotides or polypeptides, as well as agonists orantagonists of t-PALP, could be used to prevent and heal damage to thelungs due to various pathological states. A growth factor such as t-PALPpolynucleotides or polypeptides, as well as agonists or antagonists oft-PALP, which could stimulate proliferation and differentiation andpromote the repair of alveoli and brochiolar epithelium to prevent ortreat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treatedusing t-PALP polynucleotides or polypeptides, agonists or antagonists oft-PALP. Also, t-PALP polynucleotides or polypeptides, as well asagonists or antagonists of t-PALP, could be used to stimulate theproliferation of and differentiation of type II pneumocytes, which mayhelp treat or prevent disease such as hyaline membrane diseases, such asinfant respiratory distress syndrome and bronchopulmonary displasia, inpremature infants.

t-PALP polynucleotides or polypeptides, as well as agonists orantagonists of t-PALP, could stimulate the proliferation anddifferentiation of hepatocytes and, thus, could be used to alleviate ortreat liver diseases and pathologies such as fulminant liver failurecaused by cirrhosis, liver damage caused by viral hepatitis and toxicsubstances (i.e., acetaminophen, carbon tetraholoride and otherhepatotoxins known in the art).

In addition, t-PALP polynucleotides or polypeptides, as well as agonistsor antagonists of t-PALP, could be used treat or prevent the onset ofdiabetes mellitus. In patients with newly diagnosed Types I and IIdiabetes, where some islet cell function remains, t-PALP polynucleotidesor polypeptides, as well as agonists or antagonists of t-PALP, could beused to maintain the islet function so as to alleviate, delay or preventpermanent manifestation of the disease. Also, t-PALP polynucleotides orpolypeptides, as well as agonists or antagonists of t-PALP, could beused as an auxiliary in islet cell transplantation to improve or promoteislet cell function.

Neurological Diseases

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing polynucleotides or polypeptides, aswell as agonists or antagonists of the present invention, fortherapeutic purposes, for example, to stimulate neurological cellproliferation and/or differentiation. Therefore, polynucleotides,polypeptides, agonists and/or antagonists of the invention may be usedto treat and/or detect neurologic diseases. Moreover, polynucleotides orpolypeptides, or agonists or antagonists of the invention, can be usedas a marker or detector of a particular nervous system disease ordisorder.

Examples of neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include brain diseases, such as metabolic braindiseases which includes phenylketonuria such as maternalphenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenasecomplex deficiency, Wernicke's Encephalopathy, brain edema, brainneoplasms such as cerebellar neoplasms which include infratentorialneoplasms, cerebral ventricle neoplasms such as choroid plexusneoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavandisease, cerebellar diseases such as cerebellar ataxia which includespinocerebellar degeneration such as ataxia telangiectasia, cerebellardyssynergia, Friederich's Ataxia, Machado-Joseph Disease,olivopontocerebellar atrophy, cerebellar neoplasms such asinfratentorial neoplasms, diffuse cerebral sclerosis such asencephalitis periaxialis, globoid cell leukodystrophy, metachromaticleukodystrophy and subacute sclerosing panencephalitis, cerebrovasculardisorders (such as carotid artery diseases which include carotid arterythrombosis, carotid stenosis and Moyamoya Disease, cerebral amyloidangiopathy, cerebral aneurysm, cerebral anoxia, cerebralarteriosclerosis, cerebral arteriovenous malformations, cerebral arterydiseases, cerebral embolism and thrombosis such as carotid arterythrombosis, sinus thrombosis and Wallenberg's Syndrome, cerebralhemorrhage such as epidural hematoma, subdural hematoma and subarachnoidhemorrhage, cerebral infarction, cerebral ischemia such as transientcerebral ischemia, Subclavian Steal Syndrome and vertebrobasilarinsufficiency, vascular dementia such as multi-infarct dementia,periventricular leukomalacia, vascular headache such as clusterheadache, migraine, dementia such as AIDS Dementia Complex, preseniledementia such as Alzheimer's Disease and Creutzfeldt-Jakob Syndrome,senile dementia such as Alzheimer's Disease and progressive supranuclearpalsy, vascular dementia such as multi-infarct dementia, encephalitiswhich include encephalitis periaxialis, viral encephalitis such asepidemic encephalitis, Japanese Encephalitis, St. Louis Encephalitis,tick-borne encephalitis and West Nile Fever, acute disseminatedencephalomyelitis, meningoencephalitis such as uveomeningoencephaliticsyndrome, Postencephalitic Parkinson Disease and subacute sclerosingpanencephalitis, encephalomalacia such as periventricular leukomalacia,epilepsy such as generalized epilepsy which includes infantile spasms,absence epilepsy, myoclonic epilepsy which includes MERRF Syndrome,tonic-clonic epilepsy, partial epilepsy such as complex partialepilepsy, frontal lobe epilepsy and temporal lobe epilepsy,post-traumatic epilepsy, status epilepticus such as Epilepsia PartialisContinua, Hallervorden-Spatz Syndrome, hydrocephalus such asDandy-Walker Syndrome and normal pressure hydrocephalus, hypothalamicdiseases such as hypothalamic neoplasms, cerebral malaria, narcolepsywhich includes cataplexy, bulbar poliomyelitis, cerebri pseudotumor,Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebraltoxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, centralnervous system infections such as AIDS Dementia Complex, Brain Abscess,subdural empyema, encephalomyelitis such as Equine Encephalomyelitis,Venezuelan Equine Encephalomyelitis, Necrotizing HemorrhagicEncephalomyelitis, Visna, cerebral malaria, meningitis such asarachnoiditis, aseptic meningtitis such as viral meningtitis whichincludes lymphocytic choriomeningitis. Bacterial meningtitis whichincludes Haemophilus Meningtitis, Listeria Meningtitis, MeningococcalMeningtitis such as Waterhouse-Friderichsen Syndrome, PneumococcalMeningtitis and meningeal tuberculosis, fungal meningitis such asCryptococcal Meningtitis, subdural effusion, meningoencephalitis such asuvemeningoencephalitic syndrome, myelitis such as transverse myelitis,neurosyphilis such as tabes dorsalis, poliomyelitis which includesbulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases(such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy,Gerstmann-Straussler Syndrome, Kuru, Scrapie) cerebral toxoplasmosis,central nervous system neoplasms such as brain neoplasms that includecerebellear neoplasms such as infratentorial neoplasms, cerebralventricle neoplasms such as choroid plexus neoplasms, hypothalamicneoplasms and supratentorial neoplasms, meningeal neoplasms, spinal cordneoplasms which include epidural neoplasms, demyelinating diseases suchas Canavan Diseases, diffuse cerebral sceloris which includesadrenoleukodystrophy, encephalitis periaxialis, globoid cellleukodystrophy, diffuse cerebral sclerosis such as metachromaticleukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagicencephalomyelitis, progressive multifocal leukoencephalopathy, multiplesclerosis, central pontine myelinolysis, transverse myelitis,neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue Syndrome,Visna, High Pressure Nervous Syndrome, Meningism, spinal cord diseasessuch as amyotonia congenita, amyotrophic lateral sclerosis, spinalmuscular atrophy such as Werdnig-Hoffiann Disease, spinal cordcompression, spinal cord neoplasms such as epidural neoplasms,syringomyelia, Tabes Dorsalis, Stiff-Man Syndrome, mental retardationsuch as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome,Down Syndrome, Gangliosidoses such as gangliosidoses G(M1), SandhoffDisease, Tay-Sachs Disease, Hartnup Disease, homocystinuria,Laurence-Moon-Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup UrineDisease, mucolipidosis such as fucosidosis, neuronalceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria suchas maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome,Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome, nervoussystem abnormalities such as holoprosencephaly, neural tube defects suchas anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity,encephalocele, meningocele, meningomyelocele, spinal dysraphism such asspina bifida cystica and spina bifida occulta, hereditary motor andsensory neuropathies which include Charcot-Marie Disease, Hereditaryoptic atrophy, Refsum's Disease, hereditary spastic paraplegia,Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathiessuch as Congenital Analgesia and Familial Dysautonomia, Neurologicmanifestations (such as agnosia that include Gerstmann's Syndrome,Amnesia such as retrograde amnesia, apraxia, neurogenic bladder,cataplexy, communicative disorders such as hearing disorders thatincludes deafness, partial hearing loss, loudness recruitment andtinnitus, language disorders such as aphasia which include agraphia,anomia, broca aphasia, and Wernicke Aphasia, Dyslexia such as AcquiredDyslexia, language development disorders, speech disorders such asaphasia which includes anomia, broca aphasia and Wernicke Aphasia,articulation disorders, communicative disorders such as speech disorderswhich include dysarthria, echolalia, mutism and stuttering, voicedisorders such as aphonia and hoarseness, decerebrate state, delirium,fasciculation, hallucinations, meningism, movement disorders such asangelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia,muscle hypotonia, myoclonus, tic, torticollis and tremor, musclehypertonia such as muscle rigidity such as stiff-man syndrome, musclespasticity, paralysis such as facial paralysis which includes HerpesZoster Oticus, Gastroparesis, Hemiplegia, ophthalmoplegia such asdiplopia, Duane's Syndrome, Horner's Syndrome, Chronic progressiveexternal ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis,Tropical Spastic Paraparesis, Paraplegia such as Brown-Sequard Syndrome,quadriplegia, respiratory paralysis and vocal cord paralysis, paresis,phantom limb, taste disorders such as ageusia and dysgeusia, visiondisorders such as amblyopia, blindness, color vision defects, diplopia,hemianopsia, scotoma and subnormal vision, sleep disorders such ashypersomnia which includes Kleine-Levin Syndrome, insomnia, andsomnambulism, spasm such as trismus, unconsciousness such as coma,persistent vegetative state and syncope and vertigo, neuromusculardiseases such as amyotonia congenita, amyotrophic lateral sclerosis,Lambert-Eaton Myasthenic Syndrome, motor neuron disease, muscularatrophy such as spinal muscular atrophy, Charcot-Marie Disease andWerdnig-Hoffmann Disease, Postpoliomyelitis Syndrome, MuscularDystrophy, Myasthenia Gravis, Myotonia Atrophica, Myotonia Confenita,Nemaline Myopathy, Familial Periodic Paralysis, MultiplexParamyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome,peripheral nervous system diseases such as acrodynia, amyloidneuropathies, autonomic nervous system diseases such as Adie's Syndrome,Barre-Lieou Syndrome, Familial Dysautonomia, Horner's Syndrome, ReflexSympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseasessuch as Acoustic Nerve Diseases such as Acoustic Neuroma which includesNeurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia,Melkersson-Rosenthal Syndrome, ocular motility disorders which includesamblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia suchas Duane's Syndrome, Horner's Syndrome, Chronic Progressive ExternalOphthalmoplegia which includes Kearns Syndrome, Strabismus such asEsotropia and Exotropia, Oculomotor Nerve Paralysis, Optic NerveDiseases such as Optic Atrophy which includes Hereditary Optic Atrophy,Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica,Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, DemyelinatingDiseases such as Neuromyelitis Optica and Swayback, Diabeticneuropathies such as diabetic foot, nerve compression syndromes such ascarpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndromesuch as cervical rib syndrome, ulnar nerve compression syndrome,neuralgia such as causalgia, cervico-brachial neuralgia, facialneuralgia and trigeminal neuralgia, neuritis such as experimentalallergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritisand radiculities such as polyradiculitis, hereditary motor and sensoryneuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy,Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-HoffmannDisease, Hereditary Sensory and Autonomic Neuropathies which includeCongenital Analgesia and Familial Dysautonomia, POEMS Syndrome,Sciatica, Gustatory Sweating and Tetany).

Infectious Disease

t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, can be used to treat or detect infectious agents. For example,by increasing the immune response, particularly increasing theproliferation and differentiation of B and/or T cells, infectiousdiseases may be treated. The immune response may be increased by eitherenhancing an existing immune response, or by initiating a new immuneresponse. Alternatively, t-PALP polynucleotides or polypeptides, oragonists or antagonists of t-PALP, may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated or detected by a polynucleotide orpolypeptide and/or agonist or antagonist of the present invention.Examples of viruses, include, but are not limited to Examples ofviruses, include, but are not limited to the following DNA and RNAviruses and viral families: Arbovirus, Adenoviridae, Arenaviridae,Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae,Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae(Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex,Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, andparainfluenza), Papiloma virus, Papovaviridae, Parvoviridae,Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia), Reoviridae(e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), andTogaviridae (e.g., Rubivirus). Viruses falling within these families cancause a variety of diseases or symptoms, including, but not limited to:arthritis, bronchiollitis, respiratory syncytial virus, encephalitis,eye infections (e.g., conjunctivitis, keratitis), chronic fatiguesyndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese Bencephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever,meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt'sLymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza,Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitteddiseases, skin diseases (e.g., Kaposi's, warts), and viremia.polynucleotides or polypeptides, or agonists or antagonists of theinvention, can be used to treat or detect any of these symptoms ordiseases. In specific embodiments, polynucleotides, polypeptides, oragonists or antagonists of the invention are used to treat: meningitis,Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additionalspecific embodiment polynucleotides, polypeptides, or agonists orantagonists of the invention are used to treat patients nonresponsive toone or more other commercially available hepatitis vaccines. In afurther specific embodiment polynucleotides, polypeptides, or agonistsor antagonists of the invention are used to treat AIDS.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated or detected by a polynucleotide or polypeptideand/or agonist or antagonist of the present invention include, but notlimited to, include, but not limited to, the following Gram-Negative andGram-positive bacteria and bacterial families and fungi: Actinomycetales(e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcusneoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium),Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borreliaburgdorferi, Brucellosis, Candidiasis, Campylobacter,Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g.,Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g.,Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fungalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat or detect any ofthese symptoms or diseases. In specific embodiments, Ppolynucleotides,polypeptides, agonists or antagonists of the invention are used totreat: tetanus, Diptheria, botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated or detected by a polynucleotide or polypeptide and/or agonist orantagonist of the present invention include, but not limited to, thefollowing families or class: Amebiasis, Babesiosis, Coccidiosis,Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis,Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), malaria, pregnancycomplications, and toxoplasmosis. polynucleotides or polypeptides, oragonists or antagonists of the invention, can be used to treat or detectany of these symptoms or diseases. In specific embodiments,polynucleotides, polypeptides, or agonists or antagonists of theinvention are used to treat malaria.

Preferably, treatment using a polypeptide or polynucleotide and/oragonist or antagonist of the present invention could either be byadministering an effective amount of a polypeptide to the patient, or byremoving cells from the patient, supplying the cells with apolynucleotide of the present invention, and returning the engineeredcells to the patient (ex vivo therapy). Moreover, the polypeptide orpolynucleotide of the present invention can be used as an antigen in avaccine to raise an immune response against infectious disease.

Regeneration

t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, can be used to differentiate, proliferate, and attract cells,leading to the regeneration of tissues. (See, Science 276:59–87 (1997).)The regeneration of tissues could be used to repair, replace, or protecttissue damaged by congenital defects, trauma (wounds, burns, incisions,or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vasculature (including vascularand lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, t-PALP polynucleotides or polypeptides, or agonists orantagonists of t-PALP, may increase regeneration of tissues difficult toheal. For example, increased tendon/ligament regeneration would quickenrecovery time after damage. t-PALP polynucleotides or polypeptides, oragonists or antagonists of t-PALP, of the present invention could alsobe used prophylactically in an effort to avoid damage. Specific diseasesthat could be treated include of tendinitis, carpal tunnel syndrome, andother tendon or ligament defects. A further example of tissueregeneration of non-healing wounds includes pressure ulcers, ulcersassociated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by usingt-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, to proliferate and differentiate nerve cells. Diseases thatcould be treated using this method include central and peripheralnervous system diseases, neuropathies, or mechanical and traumaticdisorders (e.g., spinal cord disorders, head trauma, cerebrovasculardisease, and stoke). Specifically, diseases associated with peripheralnerve injuries, peripheral neuropathy (e.g., resulting from chemotherapyor other medical therapies), localized neuropathies, and central nervoussystem diseases (e.g., Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis, and Shy-Dragersyndrome), could all be treated using the t-PALP polynucleotides orpolypeptides, or agonists or antagonists of t-PALP.

Chemotaxis

t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, may have chemotaxis activity. A chemotaxic molecule attracts ormobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells,mast cells, eosinophils, epithelial and/or endothelial cells) to aparticular site in the body, such as inflammation, infection, or site ofhyperproliferation. The mobilized cells can then fight off and/or healthe particular trauma or abnormality.

t-PALP polynucleotides or polypeptides, or agonists or antagonists oft-PALP, may increase chemotaxic activity of particular cells. Thesechemotactic molecules can then be used to treat inflammation, infection,hyperproliferative disorders, or any immune system disorder byincreasing the number of cells targeted to a particular location in thebody. For example, chemotaxic molecules can be used to treat wounds andother trauma to tissues by attracting immune cells to the injuredlocation. Chemotactic molecules of the present invention can alsoattract fibroblasts, which can be used to treat wounds.

It is also contemplated that t-PALP polynucleotides or polypeptides, oragonists or antagonists of t-PALP, may inhibit chemotactic activity.These molecules could also be used to treat disorders. Thus, t-PALPpolynucleotides or polypeptides, or agonists or antagonists of t-PALP,could be used as an inhibitor of chemotaxis.

Binding Activity

t-PALP polypeptides may be used to screen for molecules that bind tot-PALP or for molecules to which t-PALP binds. The binding of t-PALP andthe molecule may activate (agonist), increase, inhibit (antagonist), ordecrease activity of the t-PALP or the molecule bound. Examples of suchmolecules include antibodies, oligonucleotides, proteins (e.g.,receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand oft-PALP, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, themolecule can be closely related to the natural receptor to which t-PALPbinds, or at least, a fragment of the receptor capable of being bound byt-PALP (e.g., active site). In either case, the molecule can berationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express t-PALP, either as a secreted protein oron the cell membrane. Preferred cells include cells from mammals, yeast,Drosophila, or E. coli. Cells expressing t-PALP(or cell membranecontaining the expressed polypeptide) are then preferably contacted witha test compound potentially containing the molecule to observe binding,stimulation, or inhibition of activity of either t-PALP or the molecule.

The assay may simply test binding of a candidate compound tot-PALP,wherein binding is detected by a label, or in an assay involvingcompetition with a labeled competitor. Further, the assay may testwhether the candidate compound results in a signal generated by bindingto t-PALP.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining t-PALP, measuring t-PALP/molecule activity or binding, andcomparing the t-PALP/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure t-PALP level or activity in asample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure t-PALP level or activity by eitherbinding, directly or indirectly, to t-PALP or by competing with t-PALPfor a substrate.

Additionally, the receptor to which t-PALP binds can be identified bynumerous methods known to those of skill in the art, for example, ligandpanning and FACS sorting (Coligan, et al., Current Protocols in Immun.,1(2), Chapter 5, (1991)). For example, expression cloning is employedwherein polyadenylated RNA is prepared from a cell responsive to thepolypeptides, for example, NIH3T3 cells which are known to containmultiple receptors for the FGF family proteins, and SC-3 cells, and acDNA library created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to thepolypeptides. Transfected cells which are grown on glass slides areexposed to the polypeptide of the present invention, after they havebeen labelled. The polypeptides can be labeled by a variety of meansincluding iodination or inclusion of a recognition site for asite-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of t-PALP therebyeffectively generating agonists and antagonists of t-PALP. Seegenerally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252,and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol.8:724–33 (1997); Harayama, S. Trends Biotechnol. 16(2):76–82 (1998);Hansson, L. O., et al., J. Mol. Biol. 287:265–76 (1999); and Lorenzo, M.M. and Blasco, R. Biotechniques 24(2):308–13 (1998) (each of thesepatents and publications are hereby incorporated by reference). In oneembodiment, alteration of t-PALP polynucleotides and correspondingpolypeptides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments into a desired t-PALP moleculeby homologous, or site-specific, recombination. In another embodiment,t-PALP polynucleotides and corresponding polypeptides may be alterred bybeing subjected to random mutagenesis by error-prone PCR, randomnucleotide insertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of t-PALP may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules. In preferred embodiments, the heterologousmolecules are t-PA and related protease-like molecules which possesssuch functions as the activation of plasminogen family members. Infurther preferred embodiments, the heterologous molecule is a growthfactor such as, for example, platelet-derived growth factor (PDGF),insulin-like growth factor (IGF-I), transforming growth factor(TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor(FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5,BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2,dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active t-PALP fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the t-PALP polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and ³[H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of 3 [H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of ³ [H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the t-PALP receptor is measured and the ability of thecompound to bind to the receptor and elicit a second messenger responseis measured to determine if the compound is a potential agonist orantagonist. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting thepolypeptide/molecule. Moreover, the assays can discover agents which mayinhibit or enhance the production of the polypeptides of the inventionfrom suitably manipulated cells or tissues. Therefore, the inventionincludes a method of identifying compounds which bind to t-PALPcomprising the steps of: (a) incubating a candidate binding compoundwith t-PALP; and (b) determining if binding has occurred. Moreover, theinvention includes a method of identifying agonists/antagonistscomprising the steps of: (a) incubating a candidate compound witht-PALP, (b) assaying a biological activity, and (b) determining if abiological activity of t-PALP has been altered.

Also, one could identify molecules bind t-PALP experimentally by usingthe beta-pleated sheet regions disclosed in FIG. 3 and Table 1.Accordingly, specific embodiments of the invention are directed topolynucleotides encoding polypeptides which comprise, or alternativelyconsist of, the amino acid sequence of each beta pleated sheet regionsdisclosed in FIG. 3/Table 1. Additional embodiments of the invention aredirected to polynucleotides encoding t-PALP polypeptides which comprise,or alternatively consist of, any combination or all of the beta pleatedsheet regions disclosed in FIG. 3/Table 1. Additional preferredembodiments of the invention are directed to polypeptides whichcomprise, or alternatively consist of, the t-PALP amino acid sequence ofeach of the beta pleated sheet regions disclosed in FIG. 3/Table 1.Additional embodiments of the invention are directed to t-PALPpolypeptides which comprise, or alternatively consist of, anycombination or all of the beta pleated sheet regions disclosed in FIG.3/Table 1.

Targeted Delivery

In another embodiment, the invention provides a method of deliveringcompositions to targeted cells expressing a receptor for a polypeptideof the invention, or cells expressing a cell bound form of a polypeptideof the invention.

As discussed herein, polypeptides or antibodies of the invention may beassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions. In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering polypeptides of the invention (including antibodies) thatare associated with heterologous polypeptides or nucleic acids. In oneexample, the invention provides a method for delivering a therapeuticprotein into the targeted cell. In another example, the inventionprovides a method for delivering a single stranded nucleic acid (e.g.,antisense or ribozymes) or double stranded nucleic acid (e.g., DNA thatcan integrate into the cell's genome or replicate episomally and thatcan be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the presentinvention, or the polynucleotides encoding these polypeptides, to screenfor molecules which modify the activities of the polypeptides of thepresent invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs orany other agents which affect activities mediated by the polypeptides ofthe present invention. These methods comprise contacting such an agentwith a polypeptide of the present invention or a fragment thereof andassaying for the presence of a complex between the agent and thepolypeptide or a fragment thereof, by methods well known in the art. Insuch a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the polypeptides ofthe present invention, and is described in great detail in EuropeanPatent Application 84/03564, published on Sep. 13, 1984, which isincorporated herein by reference herein. Briefly stated, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with polypeptides of the present invention andwashed. Bound polypeptides are then detected by methods well known inthe art. Purified polypeptides are coated directly onto plates for usein the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding polypeptidesof the present invention specifically compete with a test compound forbinding to the polypeptides or fragments thereof. In this manner, theantibodies are used to detect the presence of any peptide which sharesone or more antigenic epitopes with a polypeptide of the invention.

Antisense And Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ IDNO:1, or the complementary strand thereof, and/or to nucleotidesequences contained in the deposited clone 209023. In one embodiment,antisense sequence is generated internally, by the organism, in anotherembodiment, the antisense sequence is separately administered (see, forexample, O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotidesas Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Antisense technology can be used to control gene expressionthrough antisense DNA or RNA, or through triple-helix formation.Antisense techniques are discussed for example, in Okano, J., Neurochem.56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs toinhibit the growth of the non-lymphocytic leukemia cell line HL-60 andother cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcOR1 site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the t-PALP antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the t-PALP antisense nucleic acid. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding t-PALP, or fragments thereof, can beby any promoter known in the art to act in vertebrate, preferably humancells. Such promoters can be inducible or constitutive. Such promotersinclude, but are not limited to, the SV40 early promoter region(Bernoist and Chambon, Nature 29:304–310 (1981), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al.,Cell 22:787–797 (1980), the herpes thymidine promoter (Wagner et al.,Proc. Natl. Acad. Sci. U.S.A. 78:1441–1445 (1981), the regulatorysequences of the metallothionein gene (Brinster, et al., Nature296:39–42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a t-PALPgene. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded t-PALP antisense nucleic acids, a single strandof the duplex DNA may thus be tested, or triplex formation may beassayed. The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid. Generally,the larger the hybridizing nucleic acid, the more base mismatches with at-PALP RNA it may contain and still form a stable duplex (or triplex asthe case may be). One skilled in the art can ascertain a tolerabledegree of mismatch by use of standard procedures to determine themelting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333–335. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of t-PALP shown in FIGS. 1A–Bcould be used in an antisense approach to inhibit translation ofendogenous t-PALP mRNA. Oligonucleotides complementary to the 5′untranslated region of the mRNA should include the complement of the AUGstart codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′- or coding region of t-PALP mRNA, antisense nucleic acids shouldbe at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides or at least 50nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553–6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648–652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958–976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539–549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625–6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131–6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327–330).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448–7451), etc.

While antisense nucleotides complementary to the t-PALP coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222–1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy t-PALP mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of t-PALP (FIGS. 1A–B). Preferably, the ribozymeis engineered so that the cleavage recognition site is located near the5′ end of the t-PALP mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express t-PALPin vivo. DNA constructs encoding the ribozyme may be introduced into thecell in the same manner as described above for the introduction ofantisense encoding DNA. A preferred method of delivery involves using aDNA construct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous t-PALP messages and inhibit translation.Since ribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth ofscar tissue during wound healing.

The antagonist/agonist may also be employed to treat the diseasesdescribed herein.

Thus, the invention provides a method of treating disorders or diseases,including but not limited to the disorders or diseases listed throughoutthis application, associated with overexpression of a polynucleotide ofthe present invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present invention, and/or(b) a ribozyme directed to the polynucleotide of the present invention.

Other Activities

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention, as a result of the ability to stimulate vascular endothelialcell growth, may be employed in treatment for stimulatingre-vascularization of ischemic tissues due to various disease conditionssuch as thrombosis, arteriosclerosis, and other cardiovascularconditions. The polypeptide, polynucleotide, agonist, or antagonist ofthe present invention may also be employed to stimulate angiogenesis andlimb regeneration, as discussed above.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed for treating wounds due to injuries,burns, post-operative tissue repair, and ulcers since they are mitogenicto various cells of different origins, such as fibroblast cells andskeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed stimulate neuronal growth and to treatand prevent neuronal damage which occurs in certain neuronal disordersor neuro-degenerative conditions such as Alzheimer's disease,Parkinson's disease, and AIDS-related complex. A polypeptide,polynucleotide, agonist, or antagonist of the present invention may havethe ability to stimulate chondrocyte growth, therefore, they may beemployed to enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may be also be employed to prevent skin aging due to sunburnby stimulating keratinocyte growth.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed for preventing hair loss, since FGFfamily members activate hair-forming cells and promotes melanocytegrowth. Along the same lines, a polypeptide, polynucleotide, agonist, orantagonist of the present invention may be employed to stimulate growthand differentiation of hematopoietic cells and bone marrow cells whenused in combination with other cytokines.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed to maintain organs before transplantationor for supporting cell culture of primary tissues. A polypeptide,polynucleotide, agonist, or antagonist of the present invention may alsobe employed for inducing tissue of mesodermal origin to differentiate inearly embryos.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be used to modulate mammalian characteristics, suchas body height, weight, hair color, eye color, skin, percentage ofadipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery).Similarly, a polypeptide, polynucleotide, agonist, or antagonist of thepresent invention may be used to modulate mammalian metabolism affectingcatabolism, anabolism, processing, utilization, and storage of energy.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may be used to change a mammal's mental state or physicalstate by influencing biorhythms, caricadic rhythms, depression(including depressive disorders), tendency for violence, tolerance forpain, reproductive capabilities (preferably by Activin or Inhibin-likeactivity), hormonal or endocrine levels, appetite, libido, memory,stress, or other cognitive qualities.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be used as a food additive or preservative, such asto increase or decrease storage capabilities, fat content, lipid,protein, carbohydrate, vitamins, minerals, cofactors or othernutritional components.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Expression and Purification of “His-tagged” t-PALP inE. coli

The bacterial expression vector pQE9 (pD10) is used for bacterialexpression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311). pQE9 encodes ampicillin antibiotic resistance (“Ampr”)and contains a bacterial origin of replication (“ori”), an IPTGinducible promoter, a ribosome binding site (“RBS”), six codons encodinghistidine residues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that an inserted DNA fragment encodinga polypeptide expresses that polypeptide with the six His residues(i.e., a “6×His tag”) covalently linked to the amino terminus of thatpolypeptide.

The DNA sequence encoding the desired portion of the t-PALP proteincomprising the mature form of the t-PALP amino acid sequence isamplified from the deposited cDNA clone using PCR oligonucleotideprimers which anneal to the amino terminal sequences of the desiredportion of the t-PALP protein and to sequences in the depositedconstruct 3′ to the cDNA coding sequence. Additional nucleotidescontaining restriction sites to facilitate cloning in the pQE9 vectorare added to the 5′ and 3′ primer sequences, respectively.

For cloning the mature form of the t-PALP protein, the 5′ primer has thesequence 5′ GGC CGA CAT GTC TGG AGG CTG TTT CTG G 3′ (SEQ ID NO:11)containing the underlined Afl III restriction site followed by 17nucleotides of the amino terminal coding sequence of the mature t-PALPsequence in SEQ ID NO:2. One of ordinary skill in the art wouldappreciate, of course, that the point in the protein coding sequencewhere the 5′ primer begins may be varied to amplify a DNA segmentencoding any desired portion of the complete t-PALP protein shorter orlonger than the mature form of the protein. The 3′ primer has thesequence 5′ GGC GGA AGC TTA TTA GGC CCC AGG AGT CCC GGC 3′ (SEQ IDNO:12) containing the underlined Hind III restriction site followed by22 nucleotides complementary to the 3′ end of the coding sequence of thet-PALP DNA sequence in FIGS. 1A, 1B, and 1C.

The amplified t-PALP DNA fragment and the vector pQE9 are digested withAfl III and Hind III and the digested DNAs are then ligated together.Insertion of the t-PALP DNA into the restricted pQE9 vector places thet-PALP protein coding region downstream from the IPTG-inducible promoterand in-frame with an initiating AUG and the six histidine codons.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance (“Kanr”),is used in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressingt-PALP protein, is available commercially from QIAGEN, Inc., supra.Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 m (“OD600”) of between 0.4 and 0.6.Isopropyl-β-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

The cells are then stirred for 3–4 hours at 4° C. in 6M guanidine-HCl,pH 8. The cell debris is removed by centrifugation, and the supernatantcontaining the t-PALP is loaded onto a nickel-nitrilo-tri-acetic acid(“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra).Proteins with a 6×His tag bind to the Ni-NTA resin with high affinityand can be purified in a simple one-step procedure (for details see: TheQIA expressionist, 1995, QIAGEN, Inc., supra). Briefly the supernatantis loaded onto the column in 6 M guanidine-HCl, pH 8, the column isfirst washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washedwith 10 volumes of 6 M guanidine-HCl pH 6, and finally the t-PALP iseluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M–1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins can be eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

The following alternative method may be used to purify t-PALP expressedin E coli when it is present in the form of inclusion bodies. Unlessotherwise specified, all of the following steps are conducted at 4–10°C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4–10° C. and the cells are harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells ware then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000–6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2–4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the t-PALPpolypeptide-containing supernatant is incubated at 4° C. overnight toallow further GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps.

To clarify the refolded t-PALP polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 μm membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 mm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

Fractions containing the t-PALP polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the t-PALP polypeptide (determined, for instance, by 16%SDS-PAGE) are then pooled.

The resultant t-PALP polypeptide exhibits greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsare observed from Commassie blue stained 16% SDS-PAGE gel when 5 μg ofpurified protein is loaded. The purified protein is also tested forendotoxin/LPS contamination, and typically the LPS content is less than0.1 ng/ml according to LAL assays.

In an additional preferred embodiment, amino acid residues Ser-122through Asp-165 of the t-PALP sequence shown in FIGS. 1A, 1B, and 1C(which is identical to residues Ser-1 through Asp-144 of SEQ ID NO:2) isexpressed using the pHE-4 bacterial expression vector (ATCC AccessionNo. 209311). In this embodiment, residues Ser-1 through Asp-144 of thet-PALP amino acid sequence shown in SEQ ID NO:2 is expressed in E. coliusing essentially the protocol described in this Example. Apolynucleotide encoding amino acid residues Ser-1 through Asp-144 of thet-PALP amino acid sequence shown in SEQ ID NO:2 is subcloned cloned intothe 5′ Nde I and 3′ Asp 718 restriction endonuclease sites of the pHE-4bacterial expression vector. One of skill in the art could easily designappropriate primers to achieve such subcloning. For example, a 5′ and 3′primer pair which has been used successfully to generate the desiredsubclone is as follows: 5′ primer: 5′-GGC TCG CAT ATG TCT GGA GGC TGTTTC TGG GAC-3′ (SEQ ID NO:29) and 3′ primer: 5′-GCG CAT GGT ACC TTA TTAGTC CTT TTT CTC CTT GGA GTT C-3′ (SEQ ID NO:30).

Example 2 Cloning and Expression of t-PALP Protein in a BaculovirusExpression System

In this illustrative example, the plasmid shuttle vector pA2 is used toinsert the cloned DNA encoding complete protein, including its naturallyassociated secretory signal (leader) sequence, into a baculovirus toexpress the mature t-PALP protein, using standard methods as describedin Summers et al., A Manual of Methods for Baculovirus Vectors andInsect Cell Culture Procedures, Texas Agricultural Experimental StationBulletin No. 1555 (1987). This expression vector contains the strongpolyhedrin promoter of the Autographa californica nuclear polyhedrosisvirus (AcMNPV) followed by convenient restriction sites such as Bam HI,Xba I and Asp 718. The polyadenylation site of the simian virus 40(“SV40”) is used for efficient polyadenylation. For easy selection ofrecombinant virus, the plasmid contains the beta-galactosidase gene fromE. coli under control of a weak Drosophila promoter in the sameorientation, followed by the polyadenylation signal of the polyhedringene. The inserted genes are flanked on both sides by viral sequencesfor cell-mediated homologous recombination with wild-type viral DNA togenerate a viable virus that express the cloned polynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31–39 (1989).

The cDNA sequence encoding the full length t-PALP protein in thedeposited clone, including the AUG initiation codon and the naturallyassociated leader sequence shown in SEQ ID NO:2, is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene. The 5′ primer has the sequence 5′ GGC CGG GAT CCG CCA TCA TGC TGTTGG CCT GGG TAC 3′ (SEQ ID NO:13) containing the underlined Bam HIrestriction enzyme site, an efficient signal for initiation oftranslation in eukaryotic cells, as described by Kozak, M., J. Mol.Biol. 196:947–950 (1987), followed by 25 of nucleotides of the sequenceof the complete t-PALP protein shown in FIGS. 1A, 1B, and 1C, beginningwith the AUG initiation codon. The 3′ primer has the sequence 5′ GGC CGGGTA CCT TAT TAG GCC CCA GGA GTC CCG GC 3′ (SEQ ID NO:14) containing theunderlined Asp 718 restriction site followed by 24 nucleotidescomplementary to the 3′ noncoding sequence in FIGS. 1A, 1B, and 1C.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with Bam HI and Asp 718 and againis purified on a 1% agarose gel. This fragment is designated herein F1.

The plasmid is digested with the restriction enzymes Bam HI and Asp 718and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA isdesignated herein “V1”.

Fragment F1 and the dephosphorylated plasmid V1 are ligated togetherwith T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts suchas XL-1 Blue (Statagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria are identified that contain the plasmid with the human t-PALPgene by digesting DNA from individual colonies using Bam HI and Asp 718and then analyzing the digestion product by gel electrophoresis. Thesequence of the cloned fragment is confirmed by DNA sequencing. Thisplasmid is designated herein pA2t-PALP.

Five μg of the plasmid pA2t-PALP is co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413–7417 (1987). One μg of BaculoGold™ virus DNA and 5 μg of theplasmid pA2t-PALP are mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27° C. The transfection solution is then removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. Cultivation is then continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10). After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant virus is called V-t-PALP.

To verify the expression of the t-PALP gene Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus V-t-PALP at a multiplicity ofinfection (“MOI”) of about 2. If radiolabeled proteins are desired, 6hours later the medium is removed and is replaced with SF900 II mediumminus methionine and cysteine (available from Life Technologies Inc.,Rockville, Md.). After 42 hours, 5 μCi of ³⁵S-methionine and 5 μCi³⁵S-cysteine (available from Amersham) are added. The cells are furtherincubated for 16 hours and then are harvested by centrifugation. Theproteins in the supernatant as well as the intracellular proteins areanalyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe mature form of the t-PALP protein and thus the cleavage point andlength of the naturally associated secretory signal peptide.

Example 3 Cloning and Expression of t-PALP in Mammalian Cells

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, hygromycin allows theidentification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulto develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy et al., Biochem J227:277–279 (1991); Bebbington et al., Bio/Technology 10:169–175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of proteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology,438–447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart etal., Cell 41:521–530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites Bam HI, Xba I and Asp 718, facilitatethe cloning of the gene of interest. The vectors contain in addition the3′ intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

The expression plasmid, pt-PALPHA, is made by cloning a portion of thecDNA encoding the mature form of the t-PALP protein into the expressionvector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen,Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37: 767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

Example 3(a) Cloning and Expression in COS Cells

A DNA fragment encoding the complete t-PALP polypeptide is cloned intothe polylinker region of the vector so that recombinant proteinexpression is directed by the CMV promoter. The plasmid constructionstrategy is as follows. The t-PALP cDNA of the deposited clone isamplified using primers that contain convenient restriction sites, muchas described above for construction of vectors for expression of t-PALPin E. coli. Suitable primers include the following, which are used inthis example. The 5′ primer, containing the underlined Bam HI site, aKozak sequence, an AUG start codon, and 25 nucleotides of the 5′ codingregion of the complete t-PALP polypeptide, has the following sequence:5′ GGC CGG GAT CCG CCA TCA TGC TGT TGG CCT GGG TAC 3′ (SEQ ID NO:15).The 3′ primer, containing the underlined Asp 718 and 24 of nucleotidescomplementary to the 3′ coding sequence immediately before the stopcodon, has the following sequence: 5′ GGC CGG GTA CCT TAT TAG GCC CCAGGA GTC CCG GC 3′ (SEQ ID NO:16).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith Bam HI and Asp 718 and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisor other means for the presence of the fragment encoding the completet-PALP polypeptide

For expression of recombinant t-PALP, COS cells are transfected with anexpression vector, as described above, using DEAE-DEXTRAN, as described,for instance, in Sambrook et al., Molecular Cloning: a LaboratoryManual, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989).Cells are incubated under conditions for expression of t-PALP by thevector.

Expression of the t-PALP-HA fusion protein is detected by radiolabelingand immunoprecipitation, using methods described in, for example Harlowet al., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1988). To this end, two daysafter transfection, the cells are labeled by incubation in mediacontaining ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3(b) Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of t-PALP polypeptide. PlasmidpC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).The plasmid contains the mouse DHFR gene under control of the SV40 earlypromoter. Chinese hamster ovary- or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (alpha minus MEM, LifeTechnologies) supplemented with the chemotherapeutic agent methotrexate.The amplification of the DHFR genes in cells resistant to methotrexate(MTX) has been well documented (see, e.g., Alt, F. W., Kellems, R. M.,Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem. 253:1357–1370,Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107–143,Page, M. J. and Sydenham, M. A. 1991, Biotechnology 9:64–68). Cellsgrown in increasing concentrations of MTX develop resistance to the drugby overproducing the target enzyme, DHFR, as a result of amplificationof the DHFR gene. If a second gene is linked to the DHFR gene, it isusually co-amplified and over-expressed. It is known in the art thatthis approach may be used to develop cell lines carrying more than 1,000copies of the amplified gene(s). Subsequently, when the methotrexate iswithdrawn, cell lines are obtained which contain the amplified geneintegrated into one or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438–447)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521–530 (1985)).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, Xba I,and Asp718. Behind these cloning sites the plasmid contains the 3′intron and polyadenylation site of the rat preproinsulin gene. Otherhigh efficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the t-PALP polypeptide in a regulated wayin mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad.Sci. USA 89:5547–5551). For the polyadenylation of the mRNA othersignals, e.g., from the human growth hormone or globin genes can be usedas well. Stable cell lines carrying a gene of interest integrated intothe chromosomes can also be selected upon co-transfection with aselectable marker such as gpt, G418 or hygromycin. It is advantageous touse more than one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzymes Bam HI and Asp718 and then dephosphorylated using calf intestinal phosphates byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

The DNA sequence encoding the t-PALP polypeptide is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thedesired portion of the gene. The 5′ primer containing the underlined BamHI site, a Kozak sequence, an AUG start codon, and 25 nucleotides of the5′ coding region of the t-PALP polypeptide, has the following sequence:5′ GGC CGG GAT CCG CCA TCA TGC TGT TGG CCT GGG TAC 3′ (SEQ ID NO:15).The 3′ primer, containing the underlined Asp 718 and 24 of nucleotidescomplementary to the 3′ coding sequence immediately before the stopcodon as shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1), has the followingsequence: 5′ GGC CGG GTA CCT TAT TAG GCC CCA GGA GTC CCG GC 3′ (SEQ IDNO:16).

The amplified fragment is digested with the endonucleases Bam HI and Asp718 and then purified again on a 1% agarose gel. The isolated fragmentand the dephosphorylated vector are then ligated with T4 DNA ligase. E.coli HB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10–14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100–200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 4 Tissue Distribution of t-PALP mRNA Expression

Northern blot analysis was carried out to examine t-PALP gene expressionin human tissues using methods described by, among others, Sambrook etal., cited above. A cDNA probe containing the entire nucleotide sequenceof the t-PALP protein (SEQ ID NO:1) was labeled with ³²P using therediprime™ DNA labeling system (Amersham Life Science), according tomanufacturer's instructions. After labeling, the probe was purifiedusing a TE Select-D G50 spin column (5 prime-3 prime, Inc.) according tomanufacturer's recommendations. The purified labeled probe was then usedto examine various human tissues for t-PALP mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) were obtained from Clontech andwere examined with the labeled probe using ExpressHyb™ hybridizationsolution (Clontech) according to manufacturer's protocol numberPT1190-1. Following hybridization and washing, the blots were mountedand exposed to film at −70° C. overnight, and films developed accordingto standard procedures.

The Northern blot experiments described above indicated expression of2.5 kb t-PALP message in the following tissues: heart, brain, placenta,lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus,prostate, testis, ovary, small intestine, colon, and peripheral bloodleukocytes.

Example 5 Isolation of t-PALP Genomic Clones

A human genomic P1 library (Genomic Systems, Inc.) is screened by PCRusing primers selected for the cDNA sequence corresponding to SEQ IDNO:1, according to the method described in Example 1. (See also,Sambrook.)

Example 6 Chromosomal Mapping of t-PALP

An oligonucleotide primer set is designed according to the sequence atthe 5′ end of SEQ ID NO:1. This primer preferably spans about 100nucleotides. This primer set is then used in a polymerase chain reactionunder the following set of conditions: 30 seconds, 95 degree C.; 1minute, 56 degree C.; 1 minute, 70 degree C. This cycle is repeated 32times followed by one 5 minute cycle at 70 degree C. Human, mouse, andhamster DNA is used as template in addition to a somatic cell hybridpanel containing individual chromosomes or chromosome fragments (Bios,Inc). The reactions is analyzed on either 8% polyacrylamide gels or 3.5%agarose gels. Chromosome mapping is determined by the presence of anapproximately 100 bp PCR fragment in the particular somatic cell hybrid.

Example 7 Construction of N-Terminal and/or C-Terminal Deletion Mutants

The following general approach may be used to clone a N-terminal orC-terminal deletion t-PALP deletion mutant. Generally, twooligonucleotide primers of about 15–25 nucleotides are derived from thedesired 5′ and 3′ positions of a polynucleotide of SEQ ID NO:1. The 5′and 3′ positions of the primers are determined based on the desiredt-PALP polynucleotide fragment. An initiation and stop codon are addedto the 5′ and 3′ primers respectively, if necessary, to express thet-PALP polypeptide fragment encoded by the polynucleotide fragment.Preferred t-PALP polynucleotide fragments are those encoding theN-terminal and C-terminal deletion mutants disclosed above in the“Polynucleotide and Polypeptide Fragments” section of the Specification.

Additional nucleotides containing restriction sites to facilitatecloning of the t-PALP polynucleotide fragment in a desired vector mayalso be added to the 5′ and 3′ primer sequences. The t-PALPpolynucleotide fragment is amplified from genomic DNA or from thedeposited cDNA clone using the appropriate PCR oligonucleotide primersand conditions discussed herein or known in the art. The t-PALPpolypeptide fragments encoded by the t-PALP polynucleotide fragments ofthe present invention may be expressed and purified in the same generalmanner as the full length polypeptides, although routine modificationsmay be necessary due to the differences in chemical and physicalproperties between a particular fragment and full length polypeptide.

As a means of exemplifying but not limiting the present invention, thepolynucleotide encoding the t-PALP polypeptide fragment C-4 to V-63 ofSEQ ID NO:2 is amplified and cloned as follows: A 5′ primer is generatedcomprising a restriction enzyme site followed by an initiation codon inframe with the polynucleotide sequence encoding the N-terminal portionof the polypeptide fragment beginning with C-4 of SEQ ID NO:2. Acomplementary 3′ primer is generated comprising a restriction enzymesite followed by a stop codon in frame with the polynucleotide sequenceencoding C-terminal portion of the t-PALP polypeptide fragment endingwith V-63 of SEQ ID NO:2.

The amplified polynucleotide fragment and the expression vector aredigested with restriction enzymes which recognize the sites in theprimers. The digested polynucleotides are then ligated together. Thet-PALP polynucleotide fragment is inserted into the restrictedexpression vector, preferably in a manner which places the t-PALPpolypeptide fragment coding region downstream from the promoter. Theligation mixture is transformed into competent E. coli cells usingstandard procedures and as described in the Examples herein. Plasmid DNAis isolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing.

Example 8 Protein Fusions of t-PALP

t-PALP polypeptides are preferably fused to other proteins. These fusionproteins can be used for a variety of applications. For example, fusionof t-PALP polypeptides to His-tag, HA-tag, protein A, IgG domains, andmaltose binding protein facilitates purification. (See EP A 394,827;Traunecker, et al., Nature 331:84–86 (1988).) Similarly, fusion toIgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclearlocalization signals fused to t-PALP polypeptides can target the proteinto a specific subcellular localization, while covalent heterodimer orhomodimers can increase or decrease the activity of a fusion protein.Fusion proteins can also create chimeric molecules having more than onefunction. Finally, fusion proteins can increase solubility and/orstability of the fused protein compared to the non-fused protein. All ofthe types of fusion proteins described above can be made by modifyingthe following protocol, which outlines the fusion of a polypeptide to anIgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector.

For example, if pC4 (Accession No. 209646) is used, the human Fc portioncan be ligated into the BamHI cloning site. Note that the 3′ BamHI siteshould be destroyed. Next, the vector containing the human Fc portion isre-restricted with BamHI, linearizing the vector, and a t-PALPpolynucleotide is ligated into this BamHI site. Note that thepolynucleotide is cloned without a stop codon, otherwise a fusionprotein will not be produced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Human IgG Fc region: GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACA (SEQ ID NO:17)CATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGC GACTCTAGAGGAT

Example 9 Production of an Antibody

Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing t-PALP is administered to an animal to inducethe production of sera containing polyclonal antibodies. In a preferredmethod, a preparation of t-PALP protein is prepared and purified torender it substantially free of natural contaminants. Such a preparationis then introduced into an animal in order to produce polyclonalantisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or protein binding fragments thereof). Suchmonoclonal antibodies can be prepared using hybridoma technology.(Köhler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol.6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerlinget al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,pp. 563–681 (1981).) In general, such procedures involve immunizing ananimal (preferably a mouse) with t-PALP polypeptide or, more preferably,with a secreted t-PALP polypeptide-expressing cell. Such cells may becultured in any suitable tissue culture medium; however, it ispreferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56 degreeC.), and supplemented with about 10 g/l of nonessential amino acids,about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP2O), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225–232 (1981).) The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the t-PALP polypeptide.

Alternatively, additional antibodies capable of binding to t-PALPpolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the t-PALPprotein-specific antibody can be blocked byt-PALP. Such antibodiescomprise anti-idiotypic antibodies to the t-PALP protein-specificantibody and can be used to immunize an animal to induce formation offurther t-PALP protein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, secreted t-PALPprotein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).)

Isolation of Antibody Fragments Directed Against t-PALP from a Libraryof scFvs.

Naturally occuring V-genes isolated from human PBLs are constructed intoa large library of antibody fragments which contain reactivities againstt-PALP to which the donor may or may not have been exposed (see e.g.,U.S. Pat. No. 5,885,793 incorporated herein in its entirety byreference).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in WO92/01047. To rescue phage displayingantibody fragments, approximately 10⁹ E. coli harbouring the phagemidare used to inoculate 50 ml of 2×TY containing 1% glucose and 100 ug/mlof ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking.Five ml of this culture is used to innoculate 50 ml of 2×TY-AMP-GLU,2×10⁸ TU of delta gene 3 helper (M13 delta gene III, see WO92/01047) areadded and the culture incubated at 37 degree C. for 45 minutes withoutshaking and then at 37 degree C. for 45 minutes with shaking. Theculture is centrifuged at 4000 r.p.m. for 10 min. and the pelletresuspended in 2 liters of of 2×TY containing 100 ug/ml ampicillin and50 ug/ml kanamycin and grown overnight. Phage are prepared as describedin WO92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harbouring a pUC19 derivative supplying the wild typegene III protein during phage morphogenesis. The culture is incubatedfor 1 hour at 37 degree C. without shaking and then for a further hourat 37 degree C. with shaking. Cells are spun down (IEC-Centra 8, 4000revs/min for 10 min), resuspended in 300 ml 2×TY broth containing 100 ugampicillin/ml and 25 ug kanamycin/ml (2×TY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 um filter (MinisartNML; Sartorius) to give a final concentration of approximately 10¹³transducing units/ml (ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 ug/ml or 10 ug/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37 degree C. and then washed 3 times in PBS. Approximately 10¹³ TU ofphage is applied to the tube and incubated for 30 minutes at roomtemperature tumbling on an over and under turntable and then left tostand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1%Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100mM triethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37 degreeC. The E. coli are then plated on TYE plates containing 1% glucose and100 ug/ml ampicillin. The resulting bacterial library is then rescuedwith delta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (seee.g., WO92/01047) and then by sequencing.

Example 10 Production of t-PALP Protein for High-Throughput ScreeningAssays

The following protocol produces a supernatant containing t-PALPpolypeptide to be tested. This supernatant can then be used in theScreening Assays described in Examples 12–19.

First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution(1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516FBiowhittaker) for a working solution of 50 ug/ml. Add 200 ul of thissolution to each well (24 well plates) and incubate at RT for 20minutes. Be sure to distribute the solution over each well (note: a12-channel pipetter may be used with tips on every other channel).Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS(Phosphate Buffered Saline). The PBS should remain in the well untiljust prior to plating the cells and plates may be poly-lysine coated inadvance for up to two weeks.

Plate 293T cells (do not carry cells past P+20) at 2×10⁵ cells/well in0.5 ml DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose andL-glutamine (12-604F Biowhittaker))/10% heat inactivated FBS(14-503FBiowhittaker)/1× Penstrep(17-602E Biowhittaker). Let the cells growovernight.

The next day, mix together in a sterile solution basin: 300 ulLipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter,aliquot approximately 2 ug of an expression vector containing apolynucleotide insert, produced by the methods described in Examples7–8, into an appropriately labeled 96-well round bottom plate. With amulti-channel pipetter, add 50 ul of the Lipofectamine/Optimem I mixtureto each well. Pipette up and down gently to mix. Incubate at RT 15–45minutes. After about 20 minutes, use a multi-channel pipetter to add 150ul Optimem I to each well. As a control, one plate of vector DNA lackingan insert should be transfected with each set of transfections.

Preferably, the transfection should be performed by tag-teaming thefollowing tasks. By tag-teaming, hands on time is cut in half, and thecells do not spend too much time on PBS. First, person A aspirates offthe media from four 24-well plates of cells, and then person B rinseseach well with 0.5–1 ml PBS. Person A then aspirates off PBS rinse, andperson B, using a 12-channel pipetter with tips on every other channel,adds the 200 ul of DNA/Lipofectamine/Optimem I complex to the odd wellsfirst, then to the even wells, to each row on the 24-well plates.Incubate at 37 degree C. for 6 hours.

While cells are incubating, prepare appropriate media, either 1% BSA inDMEM with 1× penstrep, or HGS CHO-5 media (116.6 mg/L of CaCl₂ (anhyd);0.00130 mg/L CuSO₄-5H₂O; 0.050 mg/L of Fe(NO₃)₃-9H₂O; 0.417 mg/L ofFeSO₄-7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/L ofMgSO₄; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO₃; 62.50 mg/L ofNaH₂PO₄—H₂O; 71.02 mg/L of Na₂HPO4; 0.4320 mg/L of ZnSO₄-7H₂O; 0.002mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L ofDL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L ofLinolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid;0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L ofPluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml ofL-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂O; 6.65 mg/ml of L-AsparticAcid; 29.56 mg/ml of L-Cystine-2HCL-H₂O; 31.29 mg/ml of L-Cystine-2HCL;7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/mlof Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂O; 106.97 mg/ml ofL-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL;32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/mlof L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂O; and99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-CaPantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid;15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L ofPyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin;3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L ofVitamin B₁₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L ofSodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine;0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrincomplexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrincomplexed with Oleic Acid; 10 mg/L of Methyl-B-Cyclodextrin complexedwith Retinal Acetate. Adjust osmolarity to 327 mOsm) with 2 mm glutamineand 1× penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in IL DMEM for a10% BSA stock solution). Filter the media and collect 50 ul forendotoxin assay in 15 ml polystyrene conical.

The transfection reaction is terminated, preferably by tag-teaming, atthe end of the incubation period. Person A aspirates off thetransfection media, while person B adds 1.5 ml appropriate media to eachwell. Incubate at 37 degree C. for 45 or 72 hours depending on the mediaused: 1% BSA for 45 hours or CHO-5 for 72 hours.

On day four, using a 300 ul multichannel pipetter, aliquot 600 ul in one1 ml deep well plate and the remaining supernatant into a 2 ml deepwell. The supernatants from each well can then be used in the assaysdescribed in Examples 12–19.

It is specifically understood that when activity is obtained in any ofthe assays described below using a supernatant, the activity originatesfrom either the t-PALP polypeptide directly (e.g., as a secretedprotein) or by t-PALP inducing expression of other proteins, which arethen secreted into the supernatant. Thus, the invention further providesa method of identifying the protein in the supernatant characterized byan activity in a particular assay.

Example 11 Construction of GAS Reporter Construct

One signal transduction pathway involved in the differentiation andproliferation of cells is called the Jaks-STATs pathway. Activatedproteins in the Jaks-STATs pathway bind to gamma activation site “GAS”elements or interferon-sensitive responsive element (“ISRE”), located inthe promoter of many genes. The binding of a protein to these elementsalter the expression of the associated gene.

GAS and ISRE elements are recognized by a class of transcription factorscalled Signal Transducers and Activators of Transcription, or “STATs.”There are six members of the STATs family. Stat1 and Stat3 are presentin many cell types, as is Stat2 (as response to IFN-alpha iswidespread). Stat4 is more restricted and is not in many cell typesthough it has been found in T helper class I, cells after treatment withIL-12. Stat5 was originally called mammary growth factor, but has beenfound at higher concentrations in other cells including myeloid cells.It can be activated in tissue culture cells by many cytokines.

The STATs are activated to translocate from the cytoplasm to the nucleusupon tyrosine phosphorylation by a set of kinases known as the JanusKinase (“Jaks”) family. Jaks represent a distinct family of solubletyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinasesdisplay significant sequence similarity and are generally catalyticallyinactive in resting cells.

The Jaks are activated by a wide range of receptors summarized in theTable below. (Adapted from review by Schidler and Darnell, Ann. Rev.Biochem. 64:621–51 (1995).) A cytokine receptor family, capable ofactivating Jaks, is divided into two groups: (a) Class 1 includesreceptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15,Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share aconserved cysteine motif (a set of four conserved cysteines and onetryptophan) and a WSXWS motif (a membrane proximal region encodingTrp-Ser-Xxx-Trp-Ser (SEQ ID NO:5)).

Thus, on binding of a ligand to a receptor, Jaks are activated, which inturn activate STATs, which then translocate and bind to GAS elements.This entire process is encompassed in the Jaks-STATs signal transductionpathway.

Therefore, activation of the Jaks-STATs pathway, reflected by thebinding of the GAS or the ISRE element, can be used to indicate proteinsinvolved in the proliferation and differentiation of cells. For example,growth factors and cytokines are known to activate the Jaks-STATspathway. (See Table below.) Thus, by using GAS elements linked toreporter molecules, activators of the Jaks-STATs pathway can beidentified.

JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS (elements) or ISRE IFN familyIFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 > IFP)Il-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotrohic) + + + ? 1, 3 GAS(IRF1 > Lys6 > IFP) Il-11(Pleiotrohic) ? + ? ? 1, 3 OnM(Pleiotrohic)? + + ? 1, 3 LIF(Pleiotrohic) ? + + ? 1, 3 CNTF(Pleiotrohic) −/+ + + ?1, 3 G-CSF(Pleiotrohic) ? + ? ? 1, 3 IL-12(Pleiotrohic) + − + + 1, 3 g-Cfamily IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid) − +− + 6 GAS (IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GAS IL-9(lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15 ? +? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >> Ly6)IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growthhormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5GAS(B-CAS > IRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1,3 GAS (IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

To construct a synthetic GAS containing promoter element, which is usedin the Biological Assays described in Examples 14–15, a PCR basedstrategy is employed to generate a GAS-SV40 promoter sequence. The 5′primer contains four tandem copies of the GAS binding site found in theIRF1 promoter and previously demonstrated to bind STATs upon inductionwith a range of cytokines (Rothman et al., Immunity 1:457–468 (1994).),although other GAS or ISRE elements can be used instead. The 5′ primeralso contains 18 bp of sequence complementary to the SV40 early promotersequence and is flanked with an XhoI site. The sequence of the 5′ primeris: 5′-GCG CCT CGA GAT TTC CCC GAA ATC TAG ATT TCC CCG AAA TGA TTT CCCCGA AAT GAT TTC CCC GAA ATA TCT GCC ATC TCA ATT AG-3′ (SEQ ID NO:18)

The downstream primer is complementary to the SV40 promoter and isflanked with a Hind III site: 5′-GCG GCA AGC TTT TTG CAA AGC CTA GGC-3′(SEQ ID NO:19).

PCR amplification is performed using the SV40 promoter template presentin the B-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI/Hind III and subcloned into BLSK2-.(Stratagene.) Sequencing with forward and reverse primers confirms thatthe insert contains the following sequence:

5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGA (SEQ ID NO:20)AATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGG CCTAGGCTTTTGCAAAAAGCTT:3′.

With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2reporter construct is next engineered. Here, the reporter molecule is asecreted alkaline phosphatase, or “SEAP.” Clearly, however, any reportermolecule can be instead of SEAP, in this or in any of the otherExamples. Well known reporter molecules that can be used instead of SEAPinclude chloramphenicol acetyltransferase (CAT), luciferase, alkalinephosphatase, B-galactosidase, green fluorescent protein (GFP), or anyprotein detectable by an antibody.

The above sequence confirmed synthetic GAS-SV40 promoter element issubcloned into the pSEAP-Promoter vector obtained from Clontech usingHindIII and XhoI, effectively replacing the SV40 promoter with theamplified GAS:SV40 promoter element, to create the GAS-SEAP vector.However, this vector does not contain a neomycin resistance gene, andtherefore, is not preferred for mammalian expression systems.

Thus, in order to generate mammalian stable cell lines expressing theGAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAPvector using SalI and NotI, and inserted into a backbone vectorcontaining the neomycin resistance gene, such as pGFP-1 (Clontech),using these restriction sites in the multiple cloning site, to createthe GAS-SEAP/Neo vector. Once this vector is transfected into mammaliancells, this vector can then be used as a reporter molecule for GASbinding as described in Examples 12–13.

Other constructs can be made using the above description and replacingGAS with a different promoter sequence. For example, construction ofreporter molecules containing NF-kappaB and EGR promoter sequences aredescribed in Examples 14 and 15. However, many other promoters can besubstituted using the protocols described in these Examples. Forinstance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted,alone or in combination (e.g., GAS/NF-KappaB/EGR, GAS/NF-KappaB,II-2/NFAT, or NF-KappaB/GAS). Similarly, other cell lines can be used totest reporter construct activity, such as HELA (epithelial), HUVEC(endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), orCardiomyocyte.

Example 12 High-Throughput Screening Assay for T-cell Activity

The following protocol is used to assess T-cell activity by identifyingfactors, and determining whether supernate containing a polypeptide ofthe invention proliferates and/or differentiates T-cells. T-cellactivity is assessed using the GAS/SEAP/Neo construct produced inExample 13. Thus, factors that increase SEAP activity indicate theability to activate the Jaks-STATS signal transduction pathway. TheT-cell used in this assay is Jurkat T-cells (ATCC Accession No.TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4cells (ATCC Accession No. CRL-1582) cells can also be used.

Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In order togenerate stable cell lines, approximately 2 million Jurkat cells aretransfected with the GAS-SEAP/neo vector using DMRIE-C (LifeTechnologies)(transfection procedure described below). The transfectedcells are seeded to a density of approximately 20,000 cells per well andtransfectants resistant to 1 mg/ml genticin selected. Resistant coloniesare expanded and then tested for their response to increasingconcentrations of interferon gamma. The dose response of a selectedclone is demonstrated.

Specifically, the following protocol will yield sufficient cells for 75wells containing 200 ul of cells. Thus, it is either scaled up, orperformed in multiple to generate sufficient cells for multiple 96 wellplates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep.Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of plasmidDNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C andincubate at room temperature for 15-45 mins.

During the incubation period, count cell concentration, spin down therequired number of cells (10⁷ per transfection), and resuspend inOPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degree C. for 6hrs. After the incubation, add 10 ml of RPMI+15% serum.

The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10%serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated withsupernatants containing t-PALP polypeptides or t-PALP inducedpolypeptides as produced by the protocol described in Example 10.

On the day of treatment with the supernatant, the cells should be washedand resuspended in fresh RPMI+10% serum to a density of 500,000 cellsper ml. The exact number of cells required will depend on the number ofsupernatants being screened. For one 96 well plate, approximately 10million cells (for 10 plates, 100 million cells) are required.

Transfer the cells to a triangular reservoir boat, in order to dispensethe cells into a 96 well dish, using a 12 channel pipette. Using a 12channel pipette, transfer 200 ul of cells into each well (thereforeadding 100,000 cells per well).

After all the plates have been seeded, 50 ul of the supernatants aretransferred directly from the 96 well plate containing the supernatantsinto each well using a 12 channel pipette. In addition, a dose ofexogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10,and H11 to serve as additional positive controls for the assay.

The 96 well dishes containing Jurkat cells treated with supernatants areplaced in an incubator for 48 hrs (note: this time is variable between48–72 hrs). 35 ul samples from each well are then transferred to anopaque 96 well plate using a 12 channel pipette. The opaque platesshould be covered (using sellophene covers) and stored at −20 degree C.until SEAP assays are performed according to Example 18. The platescontaining the remaining treated cells are placed at 4 degree C. andserve as a source of material for repeating the assay on a specific wellif desired.

As a positive control, 100 Unit/ml interferon gamma can be used which isknown to activate Jurkat T cells. Over 30 fold induction is typicallyobserved in the positive control wells.

The above protocol may be used in the generation of both transient, aswell as, stable transfected cells, which would be apparent to those ofskill in the art.

Example 13 High-Throughput Screening Assay Identifying Myeloid Activity

The following protocol is used to assess myeloid activity of t-PALP bydetermining whether t-PALP proliferates and/or differentiates myeloidcells. Myeloid cell activity is assessed using the GAS/SEAP/Neoconstruct produced in Example 11. Thus, factors that increase SEAPactivity indicate the ability to activate the Jaks-STATS signaltransduction pathway. The myeloid cell used in this assay is U937, apre-monocyte cell line, although TF-1, HL60, or KG1 can be used.

To transiently transfect U937 cells with the GAS/SEAP/Neo constructproduced in Example 10, a DEAE-Dextran method (Kharbanda et. al., 1994,Cell Growth & Differentiation, 5:259–265) is used. First, harvest 2×10e⁷U937 cells and wash with PBS. The U937 cells are usually grown in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS)supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.

Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffercontaining 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mMNaCl, 5 mM KCl, 375 uM Na₂HPO₄. 7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂.Incubate at 37 degrees C. for 45 min.

Wash the cells with RPMI 1640 medium containing 10% FBS and thenresuspend in 10 ml complete medium and incubate at 37 degree C. for 36hr.

The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400ug/ml G418. The G418-free medium is used for routine growth but everyone to two months, the cells should be re-grown in 400 ug/ml G418 forcouple of passages.

These cells are tested by harvesting 1×10⁸ cells (this is enough for ten96-well plates assay) and wash with PBS. Suspend the cells in 200 mlabove described growth medium, with a final density of 5×10⁵ cells/ml.Plate 200 ul cells per well in the 96-well plate (or 1×10⁵ cells/well).

Add 50 ul of the supernatant prepared by the protocol described inExample 12. Incubate at 37 degee C for 48 to 72 hr. As a positivecontrol, 100 Unit/ml interferon gamma can be used which is known toactivate U937 cells. Over 30 fold induction is typically observed in thepositive control wells. SEAP assay the supernatant according to theprotocol described in Example 18.

Example 14 High-Throughput Screening Assay Identifying Neuronal Activity

When cells undergo differentiation and proliferation, a group of genesare activated through many different signal transduction pathways. Oneof these genes, EGR1 (early growth response gene 1), is induced invarious tissues and cell types upon activation. The promoter of EGR1 isresponsible for such induction. Using the EGR1 promoter linked toreporter molecules, activation of cells can be assessed by t-PALP.

Particularly, the following protocol is used to assess neuronal activityin PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are knownto proliferate and/or differentiate by activation with a number ofmitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growthfactor), and EGF (epidermal growth factor). The EGR1 gene expression isactivated during this treatment. Thus, by stably transfecting PC12 cellswith a construct containing an EGR promoter linked to SEAP reporter,activation of PC12 cells by t-PALP can be assessed.

The EGR/SEAP reporter construct can be assembled by the followingprotocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al.,Oncogene 6:867–871 (1991)) can be PCR amplified from human genomic DNAusing the following primers: 5′-GCG CTC GAG GGA TGA CAG CGA TAG AAC CCCGG-3′ (SEQ ID NO:21); and 5′-GCG AAG CTT CGC GAC TCC CCG GAT CCG CCTC-3′ (SEQ ID NO:22).

Using the GAS:SEAP/Neo vector produced in Example 13, EGR1 amplifiedproduct can then be inserted into this vector. Linearize theGAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing theGAS/SV40 stuffer. Restrict the EGR1 amplified product with these sameenzymes. Ligate the vector and the EGR1 promoter.

To prepare 96 well-plates for cell culture, two mls of a coatingsolution (1:30 dilution of collagen type I (Upstate Biotech Inc.Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cmplate or 50 ml per well of the 96-well plate, and allowed to air dry for2 hr.

PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker)containing 10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5%heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/mlpenicillin and 100 ug/ml streptomycin on a precoated 10 cm tissueculture dish. One to four split is done every three to four days. Cellsare removed from the plates by scraping and resuspended with pipettingup and down for more than 15 times.

Transfect the EGR/SEAP/Neo construct into PC12 using the Lipofectamineprotocol described in Example 10. EGR-SEAP/PC12 stable cells areobtained by growing the cells in 300 ug/ml G418. The G418-free medium isused for routine growth but every one to two months, the cells should bere-grown in 300 ug/ml G418 for couple of passages.

To assay for neuronal activity, a 10 cm plate with cells around 70 to80% confluent is screened by removing the old medium. Wash the cellsonce with PBS (Phosphate buffered saline). Then starve the cells in lowserum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS withantibiotics) overnight.

The next morning, remove the medium and wash the cells with PBS. Scrapeoff the cells from the plate, suspend the cells well in 2 ml low serummedium. Count the cell number and add more low serum medium to reachfinal cell density as 5×10⁵ cells/ml.

Add 200 ul of the cell suspension to each well of 96-well plate(equivalent to 1×10⁵ cells/well). Add 50 ul supernatant produced byExample 10, 37 degree C. for 48 to 72 hr. As a positive control, agrowth factor known to activate PC12 cells through EGR can be used, suchas 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold inductionof SEAP is typically seen in the positive control wells. SEAP assay thesupernatant according to Example 16.

Example 15 High-Throughput Screening Assay for T-cell Activity

NF-KappaB (Nuclear Factor Kappa B) is a transcription factor activatedby a wide variety of agents including the inflammatory cytokines IL-1and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, byexposure to LPS or thrombin, and by expression of certain viral geneproducts. As a transcription factor, NF-KappaB regulates the expressionof genes involved in immune cell activation, control of apoptosis(NF-KappaB appears to shield cells from apoptosis), B and T-celldevelopment, anti-viral and antimicrobial responses, and multiple stressresponses.

In non-stimulated conditions, NF-KappaB is retained in the cytoplasmwith I-KappaB (Inhibitor Kappa B). However, upon stimulation, I-KappaBis phosphorylated and degraded, causing NF-KappaB to shuttle to thenucleus, thereby activating transcription of target genes. Target genesactivated by NF-KappaB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1MHC.

Due to its central role and ability to respond to a range of stimuli,reporter constructs utilizing the NF-KappaB promoter element are used toscreen the supernatants produced in Example 10. Activators or inhibitorsof NF-KappaB would be useful in treating diseases. For example,inhibitors of NF-KappaB could be used to treat those diseases related tothe acute or chronic activation of NF-KappaB, such as rheumatoidarthritis.

To construct a vector containing the NF-KappaB promoter element, a PCRbased strategy is employed. The upstream primer contains four tandemcopies of the NF-KappaB binding site (GGG GAC TTT CCC) (SEQ ID NO:31),18 bp of sequence complementary to the 5′ end of the SV40 early promotersequence, and is flanked with an XhoI site: 5′-GCG GCC TCG AGG GGA CTTTCC CGG GGA CTT TCC GGG GAC TTT CCG GGA CTT TCC ATC CTG CCA TCT CAA TTAG-3′ (SEQ ID NO:23).

The downstream primer is complementary to the 3′ end of the SV40promoter and is flanked with a Hind III site: 5′-GCG GCA AGC TTT TTG CAAAGC CTA GGC-3′ (SEQ ID NO:19).

PCR amplification is performed using the SV40 promoter template presentin the pB-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI and Hind III and subcloned into BLSK2-.(Stratagene) Sequencing with the T7 and T3 primers confirms the insertcontains the following sequence: 5′-

CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTT (SEQ ID NO:24)TCCGGGACTTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAA GCTT:3′.

Next, replace the SV40 minimal promoter element present in thepSEAP2-promoter plasmid (Clontech) with this NF-KappaB/SV40 fragmentusing XhoI and HindIII. However, this vector does not contain a neomycinresistance gene, and therefore, is not preferred for mammalianexpression systems.

In order to generate stable mammalian cell lines, theNF-KappaB/SV40/SEAP cassette is removed from the above NF-KappaB/SEAPvector using restriction enzymes SalI and NotI, and inserted into avector containing neomycin resistance. Particularly, theNF-KappaB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech),replacing the GFP gene, after restricting pGFP-1 with SalI and NotI.

Once NF-KappaB/SV40/SEAP/Neo vector is created, stable Jurkat T-cellsare created and maintained according to the protocol described inExample 12. Similarly, the method for assaying supernatants with thesestable Jurkat T-cells is also described in Example 14. As a positivecontrol, exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10,and H11, with a 5–10 fold activation typically observed.

Example 16 Assay for SEAP Activity

As a reporter molecule for the assays described in Examples 12–15, SEAPactivity is assayed using the Tropix Phospho-light Kit (Cat. BP-400)according to the following general procedure. The Tropix Phospho-lightKit supplies the Dilution, Assay, and Reaction Buffers used below.

Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 ul of2.5× dilution buffer into Optiplates containing 35 ul of a supernatant.Seal the plates with a plastic sealer and incubate at 65 degree C. for30 min. Separate the Optiplates to avoid uneven heating.

Cool the samples to room temperature for 15 minutes. Empty the dispenserand prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate atroom temperature 5 min. Empty the dispenser and prime with the ReactionBuffer (see the table below). Add 50 ul Reaction Buffer and incubate atroom temperature for 20 minutes. Since the intensity of thechemiluminescent signal is time dependent, and it takes about 10 minutesto read 5 plates on luminometer, one should treat 5 plates at each timeand start the second set 10 minutes later.

Read the relative light unit in the luminometer. Set H12 as blank, andprint the results. An increase in chemiluminescence indicates reporteractivity.

Reaction Buffer Formulation: # of plates Rxn buffer diluent (ml) CSPD(ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 16 904.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 6 23125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29 1557.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 185 9.25 36190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 215 10.75 42220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 245 12.25 48250 12.5 49 255 12.75 50 260 13

Example 17 High-Throughput Screening Assay Identifying Changes in SmallMolecule Concentration and Membrane Permeability

Binding of a ligand to a receptor is known to alter intracellular levelsof small molecules, such as calcium, potassium, sodium, and pH, as wellas alter membrane potential. These alterations can be measured in anassay to identify supernatants which bind to receptors of a particularcell. Although the following protocol describes an assay for calcium,this protocol can easily be modified to detect changes in potassium,sodium, pH, membrane potential, or any other small molecule which isdetectable by a fluorescent probe.

The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) tomeasure changes in fluorescent molecules (Molecular Probes) that bindsmall molecules. Clearly, any fluorescent molecule detecting a smallmolecule can be used instead of the calcium fluorescent molecule, fluo-4(Molecular Probes, Inc.; catalog no. F-14202), used here.

For adherent cells, seed the cells at 10,000–20,000 cells/well in aCo-star black 96-well plate with clear bottom. The plate is incubated ina CO₂ incubator for 20 hours. The adherent cells are washed two times inBiotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution)leaving 100 ul of buffer after the final wash.

A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. Toload the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to eachwell. The plate is incubated at 37 degrees C. in a CO₂ incubator for 60min. The plate is washed four times in the Biotek washer with HBSSleaving 100 ul of buffer.

For non-adherent cells, the cells are spun down from culture media.Cells are re-suspended to 2–5×10⁶ cells/ml with HBSS in a 50-ml conicaltube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is addedto each ml of cell suspension. The tube is then placed in a 37 degreesC. water bath for 30–60 min. The cells are washed twice with HBSS,resuspended to 1×10⁶ cells/ml, and dispensed into a microplate, 100ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate isthen washed once in Denley CellWash with 200 ul, followed by anaspiration step to 100 ul final volume.

For a non-cell based assay, each well contains a fluorescent molecule,such as fluo-4. The supernatant is added to the well, and a change influorescence is detected.

To measure the fluorescence of intracellular calcium, the FLIPR is setfor the following parameters: (1) System gain is 300–800 mW; (2)Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is488 nm; (5) Emission is 530 mm; and (6) Sample addition is 50 ul.Increased emission at 530 nm indicates an extracellular signaling eventcaused by the a molecule, either t-PALP or a molecule induced by t-PALP,which has resulted in an increase in the intracellular Ca++concentration.

Example 18 High-Throughput Screening Assay Identifying Tyrosine KinaseActivity

The Protein Tyrosine Kinases (PTK) represent a diverse group oftransmembrane and cytoplasmic kinases. Within the Receptor ProteinTyrosine Kinase RPTK) group are receptors for a range of mitogenic andmetabolic growth factors including the PDGF, FGF, EGF, NGF, HGF andInsulin receptor subfamilies. In addition there are a large family ofRPTKs for which the corresponding ligand is unknown. Ligands for RPTKsinclude mainly secreted small proteins, but also membrane-bound andextracellular matrix proteins.

Activation of RPTK by ligands involves ligand-mediated receptordimerization, resulting in transphosphorylation of the receptor subunitsand activation of the cytoplasmic tyrosine kinases. The cytoplasmictyrosine kinases include receptor associated tyrosine kinases of thesrc-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked andcytosolic protein tyrosine kinases, such as the Jak family, members ofwhich mediate signal transduction triggered by the cytokine superfamilyof receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

Because of the wide range of known factors capable of stimulatingtyrosine kinase activity, identifying whether t-PALP or a moleculeinduced by t-PALP is capable of activating tyrosine kinase signaltransduction pathways is of interest. Therefore, the following protocolis designed to identify such molecules capable of activating thetyrosine kinase signal transduction pathways.

Seed target cells (e.g., primary keratinocytes) at a density ofapproximately 25,000 cells per well in a 96 well Loprodyne Silent ScreenPlates purchased from Nalge Nunc (Naperville, Ill.). The plates aresterilized with two 30 minute rinses with 100% ethanol, rinsed withwater and dried overnight. Some plates are coated for 2 hr with 100 mlof cell culture grade type I collagen (50 mg/ml), gelatin (2%) orpolylysine (50 mg/ml), all of which can be purchased from SigmaChemicals (St. Louis, Mo.) or 10% Matrigel purchased from BectonDickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at4 degree C. Cell growth on these plates is assayed by seeding 5,000cells/well in growth medium and indirect quantitation of cell numberthrough use of alamarBlue as described by the manufacturer AlamarBiosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers#3071 from Becton Dickinson (Bedford, Mass.) are used to cover theLoprodyne Silent Screen Plates. Falcon Microtest III cell culture platescan also be used in some proliferation experiments.

To prepare extracts, A431 cells are seeded onto the nylon membranes ofLoprodyne plates (20,000/200 ml/well) and cultured overnight in completemedium. Cells are quiesced by incubation in serum-free basal medium for24 hr. After 5–20 minutes treatment with EGF (60 ng/ml) or 50 ul of thesupernatant produced in Example 10, the medium was removed and 100 ml ofextraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100,0.1% SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and a cocktail of proteaseinhibitors (#1836170) obtained from Boeheringer Mannheim (Indianapolis,Ind.) is added to each well and the plate is shaken on a rotating shakerfor 5 minutes at 4° C. The plate is then placed in a vacuum transfermanifold and the extract filtered through the 0.45 mm membrane bottomsof each well using house vacuum. Extracts are collected in a 96-wellcatch/assay plate in the bottom of the vacuum manifold and immediatelyplaced on ice. To obtain extracts clarified by centrifugation, thecontent of each well, after detergent solubilization for 5 minutes, isremoved and centrifuged for 15 minutes at 4 degree C. at 16,000×g.

Test the filtered extracts for levels of tyrosine kinase activity.Although many methods of detecting tyrosine kinase activity are known,one method is described here.

Generally, the tyrosine kinase activity of a supernatant is evaluated bydetermining its ability to phosphorylate a tyrosine residue on aspecific substrate (a biotinylated peptide). Biotinylated peptides thatcan be used for this purpose include PSK1 (corresponding to amino acids6–20 of the cell division kinase cdc2-p34) and PSK2 (corresponding toamino acids 1–17 of gastrin). Both peptides are substrates for a rangeof tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the followingcomponents in order. First, add 10 ul of 5 uM Biotinylated Peptide, then10 ul ATP/Mg₂₊ (5 mM ATP/50 mM MgCl₂), then 10 ul of 5× Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mMEGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 ul of SodiumVanadate(1 mM), and then 5 ul of water. Mix the components gently andpreincubate the reaction mix at 30 degree C. for 2 min. Initial thereaction by adding 10 ul of the control enzyme or the filteredsupernatant.

The tyrosine kinase assay reaction is then terminated by adding 10 ul of120 mm EDTA and place the reactions on ice.

Tyrosine kinase activity is determined by transferring 50 ul aliquot ofreaction mixture to a microtiter plate (MTP) module and incubating at 37degree C. for 20 min. This allows the streptavadin coated 96 well plateto associate with the biotinylated peptide. Wash the MTP module with 300μl/well of PBS four times. Next add 75 ul of anti-phospotyrosineantibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5 u/ml))to each well and incubate at 37 degree C. for one hour. Wash the well asabove.

Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim)and incubate at room temperature for at least 5 mins (up to 30 min).Measure the absorbance of the sample at 405 nm by using ELISA reader.The level of bound peroxidase activity is quantitated using an ELISAreader and reflects the level of tyrosine kinase activity.

Example 19 High-Throughput Screening Assay Identifying PhosphorylationActivity

As a potential alternative and/or compliment to the assay of proteintyrosine kinase activity described in Example 18, an assay which detectsactivation (phosphorylation) of major intracellular signal transductionintermediates can also be used. For example, as described below oneparticular assay can detect tyrosine phosphorylation of the Erk-1 andErk-2 kinases. However, phosphorylation of other molecules, such as Raf,JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specifickinase (MuSK), RAK, Tec, and Janus, as well as any other phosphoserine,phosphotyrosine, or phosphothreonine molecule, can be detected bysubstituting these molecules for Erk-1 or Erk-2 in the following assay.

Specifically, assay plates are made by coating the wells of a 96-wellELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp,(RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBSfor 1 hr at RT. The protein G plates are then treated with 2 commercialmonoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT)(Santa Cruz Biotechnology). (To detect other molecules, this step caneasily be modified by substituting a monoclonal antibody detecting anyof the above described molecules.) After 3–5 rinses with PBS, the platesare stored at 4 degree C. until use.

A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplateand cultured overnight in growth medium. The cells are then starved for48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50ul of the supernatants obtained in Example 12 for 5–20 minutes. Thecells are then solubilized and extracts filtered directly into the assayplate.

After incubation with the extract for 1 hr at RT, the wells are againrinsed. As a positive control, a commercial preparation of MAP kinase(10 ng/well) is used in place of A431 extract. Plates are then treatedwith a commercial polyclonal (rabbit) antibody (lug/ml) whichspecifically recognizes the phosphorylated epitope of the Erk-1 andErk-2 kinases (1 hr at RT). This antibody is biotinylated by standardprocedures. The bound polyclonal antibody is then quantitated bysuccessive incubations with Europium-streptavidin and Europiumfluorescence enhancing reagent in the Wallac DELFIA instrument(time-resolved fluorescence). An increased fluorescent signal overbackground indicates a phosphorylation by t-PALP or a molecule inducedby t-PALP.

Example 20 Method of Determining Alterations in the t-PALP Gene

RNA isolated from entire families or individual patients presenting witha phenotype of interest (such as a disease) is be isolated. cDNA is thengenerated from these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:1. Suggested PCRconditions consist of 35 cycles at 95 degree C. for 30 seconds; 60–120seconds at 52–58 degree C.; and 60–120 seconds at 70 degree C., usingbuffer solutions described in Sidransky, D., et al., Science 252:706(1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons oft-PALP is also determined and genomic PCR products analyzed to confirmthe results. PCR products harboring suspected mutations in t-PALP isthen cloned and sequenced to validate the results of the directsequencing.

PCR products of t-PALP are cloned into T-tailed vectors as described inHolton, T. A. and Graham, M. W., Nucleic Acids Research, 19:1156 (1991)and sequenced with T7 polymerase (United States Biochemical). Affectedindividuals are identified by mutations in t-PALP not present inunaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in a gene corresponding to t-PALP. Genomic clones isolatedaccording to Example 4 are nick-translated with digoxigenindeoxy-uridine5′-triphosphate (Boehringer Manheim), and FISH performed as described inJohnson, Cg. et al., Methods Cell Biol. 35:73–99 (1991). Hybridizationwith the labeled probe is carried out using a vast excess of human cot-1DNA for specific hybridization to the t-PALP genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region of t-PALP (hybridized by the probe)are identified as insertions, deletions, and translocations. Theset-PALP alterations are used as a diagnostic marker for an associateddisease.

Example 21 Method of Detecting Abnormal Levels of t-PALP in a BiologicalSample

t-PALP polypeptides can be detected in a biological sample, and if anincreased or decreased level of t-PALP is detected, this polypeptide isa marker for a particular phenotype. Methods of detection are numerous,and thus, it is understood that one skilled in the art can modify thefollowing assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect t-PALP in asample, preferably a biological sample. Wells of a microtiter plate arecoated with specific antibodies to t-PALP, at a final concentration of0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal andare produced by the method described in Example 11. The wells areblocked so that non-specific binding of t-PALP to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining t-PALP. Preferably, serial dilutions of the sample should beused to validate results. The plates are then washed three times withdeionized or distilled water to remove unbounded t-PALP.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25–400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate 1 hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot t-PALP polypeptide concentration on the X-axis (log scale) andfluorescence or absorbance of the Y-axis (linear scale). Interpolate theconcentration of the t-PALP in the sample using the standard curve.

Example 22 Formulation

The invention also provides methods of treatment and/or prevention ofdiseases or disorders (such as, for example, any one or more of thediseases or disorders disclosed herein) by administration to a subjectof an effective amount of a Therapeutic. By therapeutic is meant apolynucleotides or polypeptides of the invention (including fragmentsand variants), agonists or antagonists thereof, and/or antibodiesthereto, in combination with a pharmaceutically acceptable carrier type(e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistentwith good medical practice, taking into account the clinical conditionof the individual patient (especially the side effects of treatment withthe Therapeutic alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofthe Therapeutic administered parenterally per dose will be in the rangeof about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although,as noted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the Therapeutic is typically administered at a dose rateof about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1–4 injectionsper day or by continuous subcutaneous infusions, for example, using amini-pump. An intravenous bag solution may also be employed. The lengthof treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

Therapeutics can be are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any. The term “parenteral” as usedherein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547–556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167–277 (1981), and Langer, Chem. Tech. 12:98–105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see generally, Langer, Science249:1527–1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317–327 and 353–365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688–3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci.(USA) 77:4030–4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200–800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTherapeutic.

In yet an additional embodiment, the Therapeutics of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527–1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeuticuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1–10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of theTherapeutics of the invention. Associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition, the Therapeutics may be employedin conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeuticsof the invention are administered in combination with alum. In anotherspecific embodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the Therapeutics of the invention are administered incombination with members of the TNF family. TNF, TNF-related or TNF-likemolecules that may be administered with the Therapeutics of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880), TR6(International Publication No. WO 98/30694), OPG, and neutrokine-alpha(International Publication No. WO 98/18921, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2(International Publication No. WO 98/06842), and TR12, and soluble formsCD154, CD70, and CD153.

In certain embodiments, Therapeutics of the invention are administeredin combination with antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the Therapeuticsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith Therapeutics of the invention to treat AIDS and/or to prevent ortreat HIV infection.

In other embodiments, Therapeutics of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™,and/or AZITHROMYCIN™ to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with PYRIMETHAMINE™ and/orLEUCOVORIN™ to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,Therapeutics of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In specific embodiments, Therapeutics of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the Therapeutics of the invention include,but are not limited to, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™(cyclosporin), PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate),Azathioprine, glucorticosteroids, and RAPAMUNE™ (sirolimus). In aspecific embodiment, immunosuppressants may be used to prevent rejectionof organ or bone marrow transplantation.

In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, Therapeutics of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the Therapeutics of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the Therapeuticsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compostions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the Therapeutics of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, Therapeutics of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, Therapeutics of the invention are administered incombination with Rituximab. In a further embodiment, Therapeutics of theinvention are administered with Rituxmab and CHOP, or Rituxmab and anycombination of the components of CHOP.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, Il-20, and IL- 21.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (P1GF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(P1GF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259–268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649;, Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B 186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to, LEUKNE™(SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).

In an additional embodiment, the Therapeutics of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the Therapeutics of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 23 Method of Treating Decreased Levels of t-PALP

The present invention relates to a method for treating an individual inneed of an increased level of a polypeptide of the invention in the bodycomprising administering to such an individual a composition comprisinga therapeutically effective amount of an agonist of the invention(including polypeptides of the invention). Moreover, it will beappreciated that conditions caused by a decrease in the standard ornormal expression level of t-PALP in an individual can be treated byadministering t-PALP, preferably in the secreted form. Thus, theinvention also provides a method of treatment of an individual in needof an increased level of t-PALP polypeptide comprising administering tosuch an individual a Therapeutic comprising an amount of t-PALP toincrease the activity level of t-PALP in such an individual.

For example, a patient with decreased levels of t-PALP polypeptidereceives a daily dose 0.1–100 ug/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in the secreted form.The exact details of the dosing scheme, based on administration andformulation, are provided in Example 22.

Example 24 Method of Treating Increased Levels of t-PALP

The present invention also relates to a method of treating an individualin need of a decreased level of a polypeptide of the invention in thebody comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an antagonist of theinvention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production oft-PALP. This technology is one example of a method of decreasing levelsof t-PALP polypeptide, preferably a secreted form, due to a variety ofetiologies, such as cancer.

For example, a patient diagnosed with abnormally increased levels oft-PALP is administered intravenously antisense polynucleotides at 0.5,1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeatedafter a 7-day rest period if the treatment was well tolerated. Theformulation of the antisense polynucleotide is provided in Example 22.

Example 25 Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing t-PALP polypeptides, onto a patient. Generally, fibroblastsare obtained from a subject by skin biopsy. The resulting tissue isplaced in tissue-culture medium and separated into small pieces. Smallchunks of the tissue are placed on a wet surface of a tissue cultureflask, approximately ten pieces are placed in each flask. The flask isturned upside down, closed tight and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted and thechunks of tissue remain fixed to the bottom of the flask and fresh media(e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) isadded. The flasks are then incubated at 37 degree C. for approximatelyone week.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

-   -   pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219–25 (1988)), flanked        by the long terminal repeats of the Moloney murine sarcoma        virus, is digested with EcORI and HindIII and subsequently        treated with calf intestinal phosphatase. The linear vector is        fractionated on agarose gel and purified, using glass beads.

The cDNA encoding t-PALP can be amplified using PCR primers whichcorrespond to the 5′ and 3′ end sequences respectively as set forth inExample 1. Preferably, the 5′ primer contains an EcORI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector containsproperly inserted t-PALP.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the t-PALP gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the t-PALP gene(the packagingcells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether t-PALPprotein is produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 26 Gene Therapy Using Endogenous t-PALP Gene

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous t-PALP sequence with apromoter via homologous recombination as described, for example, in U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932–8935 (1989); and Zijlstra et al., Nature 342:435–438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not expressed in the cells, or is expressedat a lower level than desired.

Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous t-PALP, flanking the promoter. The targeting sequence willbe sufficiently near the 5′ end of t-PALP so the promoter will beoperably linked to the endogenous sequence upon homologousrecombination. The promoter and the targeting sequences can be amplifiedusing PCR. Preferably, the amplified promoter contains distinctrestriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ endof the first targeting sequence contains the same restriction enzymesite as the 5′ end of the amplified promoter and the 5′ end of thesecond targeting sequence contains the same restriction site as the 3′end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenoust-PALP sequence. This results in the expression of t-PALP in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the t-PALP locus, plasmid pUC18(MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMVpromoter is amplified by PCR with an XbaI site on the 5′ end and a BamHIsite on the 3′ end. Two t-PALP non-coding sequences are amplified viaPCR: one t-PALP non-coding sequence (t-PALP fragment 1) is amplifiedwith a HindIII site at the 5′ end and an Xba site at the 3′ end; theother t-PALP non-coding sequence (t-PALP fragment 2) is amplified with aBamHI site at the 5′end and a HindIII site at the 3′end. The CMVpromoter and t-PALP fragments (1 and 2) are digested with theappropriate enzymes (CMV promoter—XbaI and BamHI; t-PALP fragment1—XbaI; t-PALP fragment 2—BamHI) and ligated together. The resultingligation product is digested with HindIII, and ligated with theHindIII-digested pUC18 plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250–300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14–20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16–24 hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 27 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) t-PALP sequences into an animal to increase ordecrease the expression of the t-PALP polypeptide. The t-PALPpolynucleotide may be operatively linked to a promoter or any othergenetic elements necessary for the expression of the t-PALP polypeptideby the target tissue. Such gene therapy and delivery techniques andmethods are known in the art, see, for example, WO90/11092, WO98/11779;U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859; Tabata H. et al. (1997)Cardiovasc. Res. 35(3):470–479, Chao J et al. (1997) Pharmacol. Res.35(6):517–522, Wolff J. A. (1997) Neuromuscul. Disord. 7(5):314–318,Schwartz B. et al. (1996) Gene Ther. 3(5):405–411, Tsurumi Y. et al.(1996) Circulation 94(12):3281–3290 (incorporated herein by reference).

The t-PALP polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The t-PALP polynucleotideconstructs can be delivered in a pharmaceutically acceptable liquid oraqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the t-PALP polynucleotides may also be delivered inliposome formulations (such as those taught in Felgner P. L. et al.(1995) Ann. NY Acad. Sci. 772:126–139 and Abdallah B. et al. (1995)Biol. Cell 85(1):1–7) which can be prepared by methods well known tothose skilled in the art.

The t-PALP polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapies techniques,one major advantage of introducing naked nucleic acid sequences intotarget cells is the transitory nature of the polynucleotide synthesis inthe cells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

The t-PALP polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked t-PALP polynucleotide injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 g/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked t-PALPpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected t-PALP polynucleotide in muscle invivo is determined as follows. Suitable t-PALP template DNA forproduction of mRNA coding for t-PALP polypeptide is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The t-PALP template DNA is injected in 0.1 ml of carrier ina 1 cc syringe through a 27 gauge needle over one minute, approximately0.5 cm from the distal insertion site of the muscle into the knee andabout 0.2 cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for t-PALP protein expression. A time course for t-PALP proteinexpression may be done in a similar fashion except that quadriceps fromdifferent mice are harvested at different times. Persistence of t-PALPDNA in muscle following injection may be determined by Southern blotanalysis after preparing total cellular DNA and HIRT supernatants frominjected and control mice. The results of the above experimentation inmice can be use to extrapolate proper dosages and other treatmentparameters in humans and other animals using t-PALP naked DNA.

Example 28 t-PALP Transgenic Animals

The t-PALP polypeptides can also be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate transgenic animals. In a specific embodiment, techniquesdescribed herein or otherwise known in the art, are used to expresspolypeptides of the invention in humans, as part of a gene therapyprotocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691–698 (1994); Carver et al., Biotechnology(NY) 11:1263–1270 (1993); Wright et al., Biotechnology (NY) 9:830–834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148–6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313–321(1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.3:1803–1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717–723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171–229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64–66 (1996); Wilmut et al., Nature 385:810–813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232–6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.

Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103–106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art. The contents of each of thedocuments recited in this paragraph is herein incorporated by referencein its entirety.

Any of the t-PALP polypeptides disclosed throughout this application canbe used to generate transgenic animals. For example, DNA encoding aminoacids M(−)21 to V63 of SEQ ID NO:2 can be inserted into a vectorcontaining a promoter, such as the actin promoter, which willubiquitously express the inserted fragment. Primers that can be used togenerate such fragments include a 5′ primer containing a BamHIrestriction site: 5′-CGA AGA GGG ATC CAT GCT GTT GGC CTG GGT ACA AGC-3′(SEQ ID NO:25) and a 3′ primer, containing an XbaI restriction site:5′-GCC GGC TCT AGA TCA GAC GTA GCA CCA GGG CCC GCG CGG G-3′ (SEQ IDNO:26). This construct will express the kringle domain of t-PALP underthe control of the actin promoter for ubiquitous expression. The regionof t-PALP included in this construct extends from M(−)21 to V63 of SEQID NO:2. Correspondingly, it would also be routine for one skilled inthe art to generate 5′ and 3′ primers to express only the proteasedomain, or other fragments, of t-PALP in transgenic animals.

Similarly, DNA encoding full length t-PALP protein, for example M(−)21to A242 of SEQ ID NO:2, can also be inserted into a vector using thefollowing primers: A 5′ primer containing a BamHI restriction site:5′-CGA AGA GGG ATC CAT GCT GTT GGC CTG GGT ACA AGC-3′ (SEQ ID NO:25) anda 3′ primer, containing an XbaI restriction site: 5′-CAC TGG TCT AGA TCAGGC CCC AGG AGT CCC GGC-3′ (SEQ ID NO:27).

Besides these two examples, other fragments of t-PALP can also beinserted into a vector to create transgenics having ubiquitousexpression.

Alternatively, polynucleotides of the invention can be inserted in avector which controls tissue specific expression through a tissuespecific promoter. For example, a construct having a transferrinpromoter would express the kringle domain of t-PALP in the liver oftransgenic animals. Therefore, DNA encoding amino acids M(−)21 to V63 ofSEQ ID NO:2 can be amplified using a 5′ primer, containing a BamHIrestriction site: 5′-CGA AGA GGG ATC CAT GCT GTT GGC CTG GGT ACA AGC-3′(SEQ ID NO:25) and a 3′ primer, containing an XbaI restriction site:5′-GCC GGC TCT AGA TCA GAC GTA GCA CCA GGG CCC GCG CGG G-3′ (SEQ IDNO:26).

Similarly, DNA encoding the full length t-PALP protein can also beinserted into a vector for tissue specific expression using thefollowing primers: A 5′ primer containing a BamHI restriction site:5′-CGA AGA GGG ATC CAT GCT GTT GGC CTG GGT ACA AGC-3′ (SEQ ID NO:25) anda 3′ primer, containing an XbaI restriction site: 5′-CAC TGG TCT AGA TCAGGC CCC AGG AGT CCC GGC-3′ (SEQ ID NO:27). In addition to expressingt-PALP in a ubiquitous or tissue specific manner in transgenic animals,it would also be routine for one skilled in the art to generateconstructs which regulate t-PALP expression by a variety of other means(for example, developmentally or chemically regulated expression).

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has occurred. The level of mRNA expression of the transgene inthe tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of t-PALP polypeptides, studying conditions and/or disordersassociated with aberrant t-PALP expression, and in screening forcompounds effective in ameliorating such conditions and/or disorders.

Example 29 t-PALP Knock-Out Animals

Endogenous t-PALP gene expression can also be reduced by inactivating or“knocking out” the t-PALP gene and/or its promoter using targetedhomologous recombination. (E.g., see Smithies et al., Nature 317:230–234(1985); Thomas & Capecchi, Cell 51:503–512 (1987); Thompson et al., Cell5:313–321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe t-PALP polypeptides. The engineered cells which express andpreferably secrete the polypeptides of the invention can be introducedinto the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Knock-out animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of t-PALP polypeptides, studying conditions and/or disordersassociated with aberrant t-PALP expression, and in screening forcompounds effective in ameliorating such conditions and/or disorders.

Example 30 Assays Detecting Stimulation or Inhibition of B cellProliferation and Differentiation

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

In Vitro Assay—Purified t-PALP protein, or truncated forms thereof, isassessed for its ability to induce activation, proliferation,differentiation or inhibition and/or death in B-cell populations andtheir precursors. The activity of t-PALP protein on purified humantonsillar B cells, measured qualitatively over the dose range from 0.1to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulationassay in which purified tonsillar B cells are cultured in the presenceof either formalin-fixed Staphylococcus aureus Cowan I (SAC) orimmobilized anti-human IgM antibody as the priming agent. Second signalssuch as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicitB cell proliferation as measured by tritiated-thymidine incorporation.Novel synergizing agents can be readily identified using this assay. Theassay involves isolating human tonsillar B cells by magnetic bead (MACS)depletion of CD3-positive cells. The resulting cell population isgreater than 95% B cells as assessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 10⁵ B-cells suspended in culture medium(RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin, 10ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of 150ul. Proliferation or inhibition is quantitated by a 20 h pulse (1uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

In Vivo Assay—BALB/c mice are injected (i.p.) twice per day with bufferonly, or 2 mg/Kg of t-PALP protein, or truncated forms thereof. Micereceive this treatment for 4 consecutive days, at which time they aresacrificed and various tissues and serum collected for analyses.Comparison of H&E sections from normal and t-PALP protein-treatedspleens identify the results of the activity of t-PALP protein on spleencells, such as the diffusion of peri-arterial lymphatic sheaths, and/orsignificant increases in the nucleated cellularity of the red pulpregions, which may indicate the activation of the differentiation andproliferation of B-cell populations. Immunohistochemical studies using aB cell marker, anti-CD45R(B220), are used to determine whether anyphysiological changes to splenic cells, such as splenic disorganization,are due to increased B-cell representation within loosely defined B-cellzones that infiltrate established T-cell regions.

Flow cytometric analyses of the spleens from t-PALP protein-treated miceis used to indicate whether t-PALP protein specifically increases theproportion of ThB+, CD45R(B220)dull B cells over that which is observedin control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andt-PALP protein-treated mice.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 31 T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of ³H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100 microliters/well of mAb toCD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnightat 4° C. (1 micrograms/ml in 0.05M bicarbonate buffer, pH 9.5), thenwashed three times with PBS. PBMC are isolated by F/H gradientcentrifugation from human peripheral blood and added to quadruplicatewells (5×10⁴/well) of mAb coated plates in RPMI containing 10% FCS andP/S in the presence of varying concentrations of t-PALP protein (totalvolume 200 microliters). Relevant protein buffer and medium alone arecontrols. After 48 hr. culture at 37° C., plates are spun for 2 min. at1000 rpm and 100 microliters of supernatant is removed and stored −20°C. for measurement of IL-2 (or other cytokines) if effect onproliferation is observed. Wells are supplemented with 100 microlitersof medium containing 0.5 microCi of ³H-thymidine and cultured at 37° C.for 18–24 hr. Wells are harvested and incorporation of ³H-thymidine usedas a measure of proliferation. Anti-CD3 alone is the positive controlfor proliferation. IL-2 (100 U/ml) is also used as a control whichenhances proliferation. Control antibody which does not induceproliferation of T cells is used as the negative controls for theeffects of t-PALP proteins.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 32 Effect of t-PALP on the Expression of MHC Class II,Costimulatory and Adhesion Molecules and Cell Differentiation ofMonocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7–10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such asTNF-alpha, causes a rapid change in surface phenotype (increasedexpression of MHC class I and II, costimulatory and adhesion molecules,downregulation of FCgammaRII, upregulation of CD83). These changescorrelate with increased antigen-presenting capacity and with functionalmaturation of the dendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1–3 days with increasing concentrations of t-PALP or LPS(positive control), washed with PBS containing 1% BSA and 0.02 mM sodiumazide, and then incubated with 1:20 dilution of appropriate FITC- orPE-labeled monoclonal antibodies for 30 minutes at 4° C. After anadditional wash, the labeled cells are analyzed by flow cytometry on aFACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendriticcells, in particular IL-12, are important in the initiation of T-celldependent immune responses. IL-12 strongly influences the development ofTh1 helper T-cell immune response, and induces cytotoxic T and NK cellfunction. An ELISA is used to measure the IL-12 release as follows.Dendritic cells (106/ml) are treated with increasing concentrations oft-PALP for 24 hours. LPS (100 ng/ml) is added to the cell culture aspositive control. Supernatants from the cell cultures are then collectedand analyzed for IL-12 content using commercial ELISA kit (e.g, R & DSystems (Minneapolis, Minn.)). The standard protocols provided with thekits are used.

Effect on the expression of MHC Class II, costimulatory and adhesionmolecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1–5 days with increasing concentrations of t-PALPor LPS (positive control), washed with PBS containing 1% BSA and 0.02 mMsodium azide, and then incubated with 1:20 dilution of appropriate FITC-or PE-labeled monoclonal antibodies for 30 minutes at 4° C. After anadditional wash, the labeled cells are analyzed by flow cytometry on aFACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules thatactivate (or alternatively, inactivate) monocytes and/or increasemonocyte survival (or alternatively, decrease monocyte survival) areknown in the art and may routinely be applied to determine whether amolecule of the invention functions as an inhibitor or activator ofmonocytes. t-PALP, agonists, or antagonists of t-PALP can be screenedusing the three assays described below. For each of these assays,Peripheral blood mononuclear cells (PBMC) are purified from single donorleukopacks (American Red Cross, Baltimore, Md.) by centrifugationthrough a Histopaque gradient (Sigma). Monocytes are isolated from PBMCby counterflow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressivelylose viability when cultured in absence of serum or other stimuli. Theirdeath results from internally regulated process (apoptosis). Addition tothe culture of activating factors, such as TNF-alpha dramaticallyimproves cell survival and prevents DNA fragmentation. Propidium iodide(PI) staining is used to measure apoptosis as follows. Monocytes arecultured for 48 hours in polypropylene tubes in serum-free medium(positive control), in the presence of 100 ng/ml TNF-alpha (negativecontrol), and in the presence of varying concentrations of the compoundto be tested. Cells are suspended at a concentration of 2×10⁶/ml in PBScontaining PI at a final concentration of 5 μg/ml, and then incubaed atroom temperature for 5 minutes before FACScan analysis. PI uptake hasbeen demonstrated to correlate with DNA fragmentation in thisexperimental paradigm.

Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of t-PALP and under the same conditions,but in the absence of t-PALP. For IL-12 production, the cells are primedovernight with IFN (100 U/ml) in presence of t-PALP. LPS (10 ng/ml) isthen added. Conditioned media are collected after 24 h and kept frozenuntil use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is thenperformed using a commercially available ELISA kit (e.g, R & D Systems(Minneapolis, Minn.)) and applying the standard protocols provided withthe kit.

Oxidative burst. Purified monocytes are plated in 96-w plate at 2–1×10⁵cell/well. Increasing concentrations of t-PALP are added to the wells ina total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamineand antibiotics). After 3 days incubation, the plates are centrifugedand the medium is removed from the wells. To the macrophage monolayers,0.2 ml per well of phenol red solution (140 mM NaCl, 10 mM potassiumphosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/mlof HRPO) is added, together with the stimulant (200 nM PMA). The platesare incubated at 37° C. for 2 hours and the reaction is stopped byadding 20 μl 1N NaOH per well. The absorbance is read at 610 nm. Tocalculate the amount of H₂O₂ produced by the macrophages, a standardcurve of a H₂O₂ solution of known molarity is performed for eachexperiment.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 33 t-PALP Biological Effects

Astrocyte and Neuronal Assays

Recombinant t-PALP, expressed in Escherichia coli and purified asdescribed above, can be tested for activity in promoting the survival,neurite outgrowth, or phenotypic differentiation of cortical neuronalcells and for inducing the proliferation of glial fibrillary acidicprotein immunopositive cells, astrocytes. The selection of corticalcells for the bioassay is based on the prevalent expression of FGF-1 andFGF-2 in cortical structures and on the previously reported enhancementof cortical neuronal survival resulting from FGF-2 treatment. Athymidine incorporation assay, for example, can be used to elucidatet-PALP's activity on these cells.

Moreover, previous reports describing the biological effects of FGF-2(basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke, P. et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012–3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of t-PALP toinduce neurite outgrowth can be compared to the response achieved withFGF-2 using, for example, a thymidine incorporation assay.

Fibroblast and Endothelial Cell Assays

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.)and maintained in growth media from Clonetics. Dermal microvascularendothelial cells are obtained from Cell Applications (San Diego,Calif.). For proliferation assays, the human lung fibroblasts and dermalmicrovascular endothelial cells can be cultured at 5,000 cells/well in a96-well plate for one day in growth medium. The cells are then incubatedfor one day in 0.1% BSA basal medium. After replacing the medium withfresh 0.1% BSA medium, the cells are incubated with the test proteinsfor 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) isadded to each well to a final concentration of 10%. The cells areincubated for 4 hr. Cell viability is measured by reading in a CytoFluorfluorescence reader. For the PGE₂ assays, the human lung fibroblasts arecultured at 5,000 cells/well in a 96-well plate for one day. After amedium change to 0.1% BSA basal medium, the cells are incubated withFGF-2 or t-PALP with or without IL-1α for 24 hours. The supernatants arecollected and assayed for PGE₂ by EIA kit (Cayman, Ann Arbor, Mich.).For the IL-6 assays, the human lung fibroblasts are cultured at 5,000cells/well in a 96-well plate for one day. After a medium change to 0.1%BSA basal medium, the cells are incubated with FGF-2 or t-PALP with orwithout IL-1α for 24 hours. The supernatants are collected and assayedfor IL-6 by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or t-PALP for 3 days inbasal medium before the addition of Alamar Blue to assess effects ongrowth of the fibroblasts. FGF-2 should show a stimulation at 10–2500ng/ml which can be used to compare stimulation with t-PALP.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to adeficiency of striatal dopamine resulting from the degeneration of thenigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released.Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP⁺ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basicFGF) has trophic activity towards nigral dopaminergic neurons (Ferrariet al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, t-PALP can be evaluated to determinewhether it has an action similar to that of FGF-2 in enhancingdopaminergic neuronal survival in vitro and it can also be tested invivo for protection of dopaminergic neurons in the striatum from thedamage associated with MPTP treatment. The potential effect of t-PALP isfirst examined in vitro in a dopaminergic neuronal cell cultureparadigm. The cultures are prepared by dissecting the midbrain floorplate from gestation day 14 Wistar rat embryos. The tissue isdissociated with trypsin and seeded at a density of 200,000 cells/cm² onpolyorthinine-laminin coated glass coverslips. The cells are maintainedin Dulbecco's Modified Eagle's medium and F12 medium containing hormonalsupplements (N1). The cultures are fixed with paraformaldehyde after 8days in vitro and are processed for tyrosine hydroxylase, a specificmarker for dopminergic neurons, immunohistochemical staining.Dissociated cell cultures are prepared from embryonic rats. The culturemedium is changed every third day and the factors are also added at thattime.

Since the dopaminergic neurons are isolated from animals at gestationday 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if t-PALP acts to prolong the survival of dopaminergicneurons, it would suggest that t-PALP may be involved in Parkinson'sDisease.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 34 The Effect of t-PALP on the Growth of Vascular EndothelialCells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at2–5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. t-PALPprotein of SEQ ID NO. 2, and positive controls, such as VEGF and basicFGF (bFGF) are added, at varying concentrations. On days 4 and 6, themedium is replaced. On day 8, cell number is determined with a CoulterCounter.

An increase in the number of HUVEC cells indicates that t-PALP mayproliferate vascular endothelial cells.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 35 Stimulatory Effect of t-PALP on the Proliferation of VascularEndothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF₁₆₅ or t-PALP in 0.5% FBS) with or withoutHeparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMSmixture (1:0.05) are added per well and allowed to incubate for 1 hourat 37° C. before measuring the absorbance at 490 nm in an ELISA platereader. Background absorbance from control wells (some media, no cells)is subtracted, and seven wells are performed in parallel for eachcondition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512–518(1994).

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 36 Inhibition of PDGF-induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h,immunocytochemistry is performed by using BrdUrd Staining Kit (ZymedLaboratories). In brief, the cells are incubated with the biotinylatedmouse anti-BrdUrd antibody at 4° C. for 2 h after being exposed todenaturing solution and then incubated with the streptavidin-peroxidaseand diaminobenzidine. After counterstaining with hematoxylin, the cellsare mounted for microscopic examination, and the BrdUrd-positive cellsare counted. The BrdUrd index is calculated as a percent of theBrdUrd-positive cells to the total cell number. In addition, thesimultaneous detection of the BrdUrd staining (nucleus) and the FITCuptake (cytoplasm) is performed for individual cells by the concomitantuse of bright field illumination and dark field-UV fluorescentillumination. See, Hayashida et al., J. Biol. Chem.6:271(36):21985–21992 (1996).

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 37 Stimulation of Endothelial Migration

This example will be used to explore the possibility that t-PALP maystimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, M D; Falk, W., etal., J. Immunological Methods 1980;33:239–247).Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um(Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for atleast 6 hours at room temperature and dried under sterile air. Testsubstances are diluted to appropriate concentrations in M199supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of thefinal dilution is placed in the lower chamber of the modified Boydenapparatus. Subconfluent, early passage (2–6) HUVEC or BMEC cultures arewashed and trypsinized for the minimum time required to achieve celldetachment. After placing the filter between lower and upper chamber,2.5×10⁵ cells suspended in 50 ul M199 containing 1% FBS are seeded inthe upper compartment. The apparatus is then incubated for 5 hours at37° C. in a humidified chamber with 5% CO₂ to allow cell migration.After the incubation period, the filter is removed and the upper side ofthe filter with the non-migrated cells is scraped with a rubberpoliceman. The filters are fixed with methanol and stained with a Giemsasolution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration isquantified by counting cells of three random high-power fields (40×) ineach well, and all groups are performed in quadruplicate.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 38 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be amediator of vascular endothelium relaxation. Thus, t-PALP activity canbe assayed by determining nitric oxide production by endothelial cellsin response to t-PALP.

Nitric oxide is measured in 96-well plates of confluent microvascularendothelial cells after 24 hours starvation and a subsequent 4 hrexposure to various levels of a positive control (such as VEGF-1) andt-PALP. Nitric oxide in the medium is determined by use of the Griessreagent to measure total nitrite after reduction of nitric oxide-derivednitrate by nitrate reductase. The effect of t-PALP on nitric oxiderelease is examined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with aNO-specific polarographic electrode connected to a NO meter (Iso-NO,World Precision Instruments Inc.) (1049). Calibration of the NO elementsis performed according to the following equation:2KNO₂+2KI+2H₂SO₄62NO+I₂+2H₂O+2K₂SO₄

The standard calibration curve is obtained by adding gradedconcentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing KI and H₂SO₄. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas (1050). The culture medium is removed and HUVECsare washed twice with Dulbecco's phosphate buffered saline. The cellsare then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) To maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×10⁶ endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96–105 (1995).

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 39 Effect of t-PALP on Cord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications' CADMEC Growth Medium and used at passage 5. Forthe in vitro angiogenesis assay, the wells of a 48-well cell cultureplate are coated with Cell Applications' Attachment Factor Medium (200ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 mg Cell Applications' Chord FormationMedium containing control buffer or t-PALP (0.1 to 100 ng/ml) and thecells are cultured for an additional 48 hr. The numbers and lengths ofthe capillary-like chords are quantitated through use of the BoeckelerVIA-170 video image analyzer. All assays are done in triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.beta-esteradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 40 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system toexamine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of t-PALP to stimulate angiogenesis in CAMcan be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and theJapanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old qualembryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chickeggs. The embryos are checked for normal development and the eggs sealedwith cellotape. They are further incubated until Day 13. Thermanoxcoverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm indiameter. Sterile and salt-free growth factors are dissolved indistilled water and about 3.3 mg/5 ml are pipetted on the disks. Afterair-drying, the inverted disks are applied on CAM. After 3 days, thespecimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsedin 0.12 M sodium cacodylate buffer. They are photographed with a stereomicroscope [Wild M8] and embedded for semi- and ultrathin sectioning asdescribed above. Controls are performed with carrier disks alone.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Using a protocol based on that of Brooks, et al. (See, Cell 79:1157–64(1994); See also, Brooks, et al., Cell 92:391–400 (1998)), the effectsof t-PALP on the growth of TSU cells were analyzed in a CAM assay. Inthis experiment, seven to ten day old eggs were candeled to locate theair sac and a window of egg shell approximately 2 cm×2 cm was openedright on the top of the CAM. Approximately 1×10⁶ freshly harvested TSUcells either transfected with t-PALP or mock-transfected (withexpression vector pcDNA3 only) were placed on the CAM. Transfectionswere performed as described in Example 51. The holes were sealed withparafilm and the eggs were placed in an incubator. Seven days later, thetumor mass on the CAM was carefully cut out and weighed. Fifteen totwenty eggs were used for each treatment and the mean +/− standard errorof tumor mass (mg/CAM) was calculated. The resulting data were subjectedto the student's t-test for statistical analysis.

In one experiment, the tumor mass associated with TSU cells transfectedwith vector only was approximately 80 mg/CAM and the tumor massassociated with TSU cells transfected with t-PALP was approximately 10mg/CAM. These results suggest that t-PALP, and agonists of t-PALP, mayplay an inhibitory role in tumor growth. Moreover, it is likely thatantagonists of t-PALP, for example, anti-t-PALP antibodies of theinvention reduce the inhibitory effect of t-PALP on tumor growth.

Example 41 Angiogenesis Assay Using a Matrigel Implant in Mouse

In vivo angiogenesis assay of t-PALP measures the ability of an existingcapillary network to form new vessels in an implanted capsule of murineextracellular matrix material (Matrigel). The protein is mixed with theliquid Matrigel at 4 degree C. and the mixture is then injectedsubcutaneously in mice where it solidifies. After 7 days, the solid“plug” of Matrigel is removed and examined for the presence of new bloodvessels. Matrigel is purchased from Becton DickinsonLabware/Collaborative Biomedical Products.

When thawed at 4 degree C. the Matrigel material is a liquid. TheMatrigel is mixed with t-PALP at 150 ng/ml at 4 degree C. and drawn intocold 3 ml syringes. Female C57Bl/6 mice approximately 8 weeks old areinjected with the mixture of Matrigel and experimental protein at 2sites at the midventral aspect of the abdomen (0.5 ml/site). After 7days, the mice are sacrificed by cervical dislocation, the Matrigelplugs are removed and cleaned (i.e., all clinging membranes and fibroustissue is removed). Replicate whole plugs are fixed in neutral buffered10% formaldehyde, embedded in paraffin and used to produce sections forhistological examination after staining with Masson's Trichrome. Crosssections from 3 different regions of each plug are processed. Selectedsections are stained for the presence of vWF. The positive control forthis assay is bovine basic FGF (150 ng/ml). Matrigel alone is used todetermine basal levels of angiogenesis.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 42 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of t-PALP on ischemia, a rabbit hindlimbischemia model is created by surgical removal of one femoral arteries asdescribed previously (Takeshita, S. et al., Am J Pathol 147:1649–1660(1995)). The excision of the femoral artery results in retrogradepropagation of thrombus and occlusion of the external iliac artery.Consequently, blood flow to the ischemic limb is dependent uponcollateral vessels originating from the internal iliac artery(Takeshita, S. et al. Am J Pathol 147:1649–1660 (1995)). An interval of10 days is allowed for post-operative recovery of rabbits anddevelopment of endogenous collateral vessels. At 10 day post-operatively(day 0), after performing a baseline angiogram, the internal iliacartery of the ischemic limb is transfected with 500 mg naked t-PALPexpression plasmid by arterial gene transfer technology using ahydrogel-coated balloon catheter as described (Riessen, R. et al. HumGene Ther. 4:749–758 (1993); Leclerc, G. et al. J. Clin. Invest. 90:936–944 (1992)). When t-PALP is used in the treatment, a single bolus of500 mg t-PALP protein or control is delivered into the internal iliacartery of the ischemic limb over a period of 1 min. through an infusioncatheter. On day 30, various parameters are measured in these rabbits:(a) BP ratio—The blood pressure ratio of systolic pressure of theischemic limb to that of normal limb; (b) Blood Flow and FlowReserve—Resting FL: the blood flow during undilated condition and MaxFL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount) and Flow Reserve is reflected by theratio of max FL: resting FL; (c) Angiographic Score—This is measured bythe angiogram of collateral vessels. A score is determined by thepercentage of circles in an overlaying grid that with crossing opacifiedarteries divided by the total number m the rabbit thigh; (d) Capillarydensity—The number of collateral capillaries determined in lightmicroscopic sections taken from hindlimbs.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 43 Effect of t-PALP on Vasodilation

Since dilation of vascular endothelium is important in reducing bloodpressure, the ability of t-PALP to affect the blood pressure inspontaneously hypertensive rats (SHR) is examined. Increasing doses (0,10, 30, 100, 300, and 900 mg/kg) of the t-PALP are administered to 13–14week old spontaneously hypertensive rats (SHR). Data are expressed asthe mean +/− SEM. Statistical analysis are performed with a pairedt-test and statistical significance is defined as p<0.05 vs. theresponse to buffer alone.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 44 Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, andfactor VIII immunohistochemistry or endothelial alkaline phosphatasereaction. t-PALP expression, during the skin ischemia, is studied usingin situ hybridization.

The study in this model is divided into three parts as follows:

-   -   a) Ischemic skin    -   b) Ischemic skin wounds    -   c) Normal wounds

The experimental protocol includes:

-   -   a) Raising a 3×4 cm, single pedicle full-thickness random skin        flap (myocutaneous flap over the lower back of the animal).    -   b) An excisional wounding (4–6 mm in diameter) in the ischemic        skin (skin-flap).    -   c) Topical treatment with t-PALP of the excisional wounds (day        0, 1, 2, 3, 4 post-wounding) at the following various dosage        ranges: 1 mg to 100 mg.    -   d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21        post-wounding for histological, immunohistochemical, and in situ        studies.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 45 Peripheral Arterial Disease Model

Angiogenic therapy using t-PALP is a novel therapeutic strategy toobtain restoration of blood flow around the ischemia in case ofperipheral arterial diseases. The experimental protocol includes:

-   -   a) One side of the femoral artery is ligated to create ischemic        muscle of the hindlimb, the other side of hindlimb serves as a        control.    -   b) t-PALP protein, in a dosage range of 20 mg–500 mg, is        delivered intravenously and/or intramuscularly 3 times (perhaps        more) per week for 2–3 weeks.    -   c) The ischemic muscle tissue is collected after ligation of the        femoral artery at 1, 2, and 3 weeks for the analysis of t-PALP        expression and histology. Biopsy is also performed on the other        side of normal muscle of the contralateral hindlimb.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 46 Ischemic Myocardial Disease Model

t-PALP is evaluated as a potent mitogen capable of stimulating thedevelopment of collateral vessels, and restructuring new vessels aftercoronary artery occlusion. Alteration of t-PALP expression isinvestigated in situ. The experimental protocol includes:

-   -   a) The heart is exposed through a left-side thoracotomy in the        rat. Immediately, the left coronary artery is occluded with a        thin suture (6-0) and the thorax is closed.    -   b) t-PALP protein, in a dosage range of 20 mg–500 mg, is        delivered intravenously and/or intramuscularly 3 times (perhaps        more) per week for 2–4 weeks.    -   c) Thirty days after the surgery, the heart is removed and        cross-sectioned for morphometric and in situ analyzes.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 47 Rat Corneal Wound Healing Model

This animal model shows the effect of t-PALP on neovascularization. Theexperimental protocol includes:

-   -   a) Making a 1–1.5 mm long incision from the center of cornea        into the stromal layer.    -   b) Inserting a spatula below the lip of the incision facing the        outer corner of the eye.    -   c) Making a pocket (its base is 1–1.5 mm form the edge of the        eye).    -   d) Positioning a pellet, containing 50 ng–5 ug of t-PALP, within        the pocket.    -   e) t-PALP treatment can also be applied topically to the corneal        wounds in a dosage range of 20 mg–500 mg (daily treatment for        five days).

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 48 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

A. Diabetic db+/db+ Mouse Model.

To demonstrate that t-PALP accelerates the healing process, thegenetically diabetic mouse model of wound healing is used. The fullthickness wound healing model in the db+/db+mouse is a wellcharacterized, clinically relevant and reproducible model of impairedwound healing. Healing of the diabetic wound is dependent on formationof granulation tissue and re-epithelialization rather than contraction(Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G.et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283–293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1–7 (1983); Leiter et al., Am. J. of Pathol. 114:46–55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221–232 (1984); Robertson et al., Diabetes29(1):60–67 (1980); Giacomelli et al., Lab Invest. 40(4):460–473 (1979);Coleman, D. L., Diabetes 31 (Suppl):1–6 (1982)). The homozygous diabeticmice develop hyperglycemia that is resistant to insulin analogous tohuman type II diabetes (Mandel et al., J. Immunol. 120:1375–1377(1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235–1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245–251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1–5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

t-PALP is administered using at a range different doses of t-PALP, from4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle controlgroups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) untreated; and 3) treatedgroup.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm², the corresponding size of the dermalpunch. Calculations are made using the following formula:[Open area on day8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with t-PALP. This assessment included verification of thepresence of cell accumulation, inflammatory cells, capillaries,fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometeris used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer can serve as a positive tissuecontrol and human brain tissue can be used as a negative tissue control.Each specimen includes a section with omission of the primary antibodyand substitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0–8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, S. M. Glucocorticoids andWound healing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280–302 (1989); Wahl, S. M. et al., J. Immunol. 115: 476–481(1975); Werb, Z. et al., J. Exp. Med. 147:1684–1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability (Ebert, R. H., et al., An. Intern. Med.37:701–705 (1952)), fibroblast proliferation, and collagen synthesis(Beck, L. S. et al., Growth Factors. 5: 295–304 (1991); Haynes, B. F. etal., J. Clin. Invest. 61: 703–797 (1978)) and producing a transientreduction of circulating monocytes (Haynes, B. F., et al., J. Clin.Invest. 61: 703–797 (1978); Wahl, S. M., “Glucocorticoids and woundhealing”, In: Antiinflammatory Steroid Action: Basic and ClinicalAspects, Academic Press, New York, pp. 280–302 (1989)). The systemicadministration of steroids to impaired wound healing is a well establishphenomenon in rats (Beck, L. S. et al., Growth Factors. 5: 295–304(1991); Haynes, B. F., et al., J. Clin. Invest. 61: 703–797 (1978);Wahl, S. M., “Glucocorticoids and wound healing”, In: AntiinflammatorySteroid Action: Basic and Clinical Aspects, Academic Press, New York,pp. 280–302 (1989); Pierce, G. F. et al., Proc. Natl. Acad. Sci. USA 86:2229–2233 (1989)).

To demonstrate that t-PALP can accelerate the healing process, theeffects of multiple topical applications of t-PALP on full thicknessexcisional skin wounds in rats in which healing has been impaired by thesystemic administration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250–300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1–5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue is no longer visible and thewound is covered by a continuous epithelium.

t-PALP is administered using at a range different doses of t-PALP, from4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle controlgroups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) t-PALP treated groups.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1is 64 mm², the corresponding size of the dermal punch. Calculations aremade using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining isperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin is improved by treatmentwith t-PALP. A calibrated lens micrometer is used by a blinded observerto determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 49 Lymphadema Animal Model

The purpose of this experimental approach is to create an appropriateand consistent lymphedema model for testing the therapeutic effects oft-PALP in lymphangiogenesis and re-establishment of the lymphaticcirculatory system in the rat hind limb. Effectiveness is measured byswelling volume of the affected limb, quantification of the amount oflymphatic vasculature, total blood plasma protein, and histopathology.Acute lymphedema is observed for 7–10 days. Perhaps more importantly,the chronic progress of the edema is followed for up to 3–4 weeks.

Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately 350 g are dosedwith Pentobarbital. Subsequently, the right legs are shaved from knee tohip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood isdrawn for serum total protein testing. Circumference and volumetricmeasurements are made prior to injecting dye into paws after marking 2measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). Theintradermal dorsum of both right and left paws are injected with 0.05 mlof 1% Evan's Blue. Circumference and volumetric measurements are thenmade following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is madecircumferentially allowing the femoral vessels to be located. Forcepsand hemostats are used to dissect and separate the skin flaps. Afterlocating the femoral vessels, the lymphatic vessel that runs along sideand underneath the vessel(s) is located. The main lymphatic vessels inthis area are then electrically coagulated or suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosisand adductors) are bluntly dissected. The popliteal lymph node is thenlocated. The 2 proximal and 2 distal lymphatic vessels and distal bloodsupply of the popliteal node are then and ligated by suturing. Thepopliteal lymph node, and any accompanying adipose tissue, is thenremoved by cutting connective tissues.

Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (A J Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5–7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effectplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limbmovement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople then those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetizedwith Pentobarbital and are tested prior to surgery. For dailyvolumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software (Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serumseparated prior to surgery and then at conclusion for total protein andCa2+ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared fortissue collection. The limbs are amputated using a quillitine, then bothexperimental and control legs are cut at the ligature and weighed. Asecond weighing is done as the tibio-cacaneal joint is disarticulatedand the foot is weighed.

Histological Preparations: The transverse muscle located behind the knee(popliteal) area is dissected and arranged in a metal mold, filled withfreezeGel, dipped into cold methylbutane, placed into labeled samplebags at −80EC until sectioning. Upon sectioning, the muscle is observedunder fluorescent microscopy for lymphatics.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 50 Suppression of TNF Alpha-Induced Adhesion Molecule Expressionby t-PALP

The recruitment of lymphocytes to areas of inflammation and angiogenesisinvolves specific receptor-ligand interactions between cell surfaceadhesion molecules (CAMs) on lymphocytes and the vascular endothelium.The adhesion process, in both normal and pathological settings, followsa multi-step cascade that involves intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelialleukocyte adhesion molecule-1 (E-selectin) expression on endothelialcells (EC). The expression of these molecules and others on the vascularendothelium determines the efficiency with which leukocytes may adhereto the local vasculature and extravasate into the local tissue duringthe development of an inflammatory response. The local concentration ofcytokines and growth factor participate in the modulation of theexpression of these CAMs.

Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine,is a stimulator of all three CAMs on endothelial cells and may beinvolved in a wide variety of inflammatory responses, often resulting ina pathological outcome.

The potential of t-PALP to mediate a suppression of TNF-a induced CAMexpression can be examined. A modified ELISA assay which uses ECs as asolid phase absorbent is employed to measure the amount of CAMexpression on TNF-a treated ECs when co-stimulated with a member of theFGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC)cultures are obtained from pooled cord harvests and maintained in growthmedium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCSand 1% penicillin/streptomycin in a 37 degree C. humidified incubatorcontaining 5% CO₂. HUVECs are seeded in 96-well plates at concentrationsof 1×10⁴ cells/well in EGM medium at 37 degree C. for 18–24 hrs or untilconfluent. The monolayers are subsequently washed 3 times with aserum-free solution of RPMI-1640 supplemented with 100 U/ml penicillinand 100 mg/ml streptomycin, and treated with a given cytokine and/orgrowth factor(s) for 24 h at 37 degree C. Following incubation, thecells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard96 well plate to confluence. Growth medium is removed from the cells andreplaced with 90 ul of 199 Medium (10% FBS). Samples for testing andpositive or negative controls are added to the plate in triplicate (in10 ul volumes). Plates are incubated at 37 degree C. for either 5 h(selectin and integrin expression) or 24 h (integrin expression only).Plates are aspirated to remove medium and 100 μl of 0.1%paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well. Platesare held at 4° C. for 30 min.

Fixative is then removed from the wells and wells are washed 1× withPBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA.

Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000dilution) to each well and incubated at 37° C. for 30 min. Wells arewashed X3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol PhosphatepNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPPsubstrate in glycine buffer is added to each test well. Standard wellsin triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000(10⁰)>10^(−0.5)>10⁻¹>10^(−1.5)0.5 μl of each dilution is added totriplicate wells and the resulting AP content in each well is 5.50 ng,1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added toeach of the standard wells. The plate must be incubated at 37° C. for 4h. A volume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

The studies described in this example tested activity in t-PALP protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of t-PALP polynucleotides (e.g., genetherapy), agonists, and/or antagonists of t-PALP.

Example 51 Effect of Conditioned Medium from t-PALP Transfectants onEndothelial Cells

Conditioned medium from TSU cells was removed following transienttransfection with a pcDNA3/t-PALP expression construct. One hundredmicrograms of the pcDNA3/t-PALP or pcDNA vector plasmid DNAs weretransfected into 1×10⁶ cells in 100 mm dishes by the CaPO4 method.Transfected cultures were maintained in 10% calf serum-DMEM (Biofluids,Rockville, Md.) containing 800 mg/ml of G418. Clones surviving the G418selection were pooled and Northern analysis was performed to confirmexpression. Aliquots of either 50, 100 or 200 microliters of theconditioned medium was added to the culture medium of endothelial cells.Treated cultures were then incubated at 37° C. The number of cells ineach culture was then determined.

Endothelial cell cultures which were treated with 50 or 100 microlitersof conditioned medium from TSU cells transfected with t-PALP exhibitedno significant effect on the number of cells in the culture. However,endothelial cell cultures which were treated with 200 microliters ofconditioned medium from TSU cells transfected with t-PALP contained lesscells than endothelial cell cultures which were treated with controlconditioned medium. In one experiment approximately 180,000 cells werecounted in endothelial cell cultures treated with control conditionedmedium as compared to 120,000 cells counted in endothelial cell culturestreated with conditioned medium from TSU cells transfected with t-PALP.

Such a decrease in endothelial cell number in cultures treated withconditioned medium from TSU cell cultures transfected with t-PALPsuggests that t-PALP may play a role in endothelial cell growth. Becauset-PALP may play a role in endothelial cell growth, it is likely thatt-PALP, agonists and/or antagonists thereof, may play a role asmediators of angiogenesis. t-PALP polypeptides and agonists of theinvention may function to inhibit angiogenesis, whereas antagonists oft-PALP, for example, anti-t-PALP antibodies of the invention, mayfunction to increase angiogenesis.

Example 52 Effect of t-PALP on Tumor Growth in Nude Mice

The ability of t-PALP to inhibit tumor growth may be assessed bymonitoring the growth rate of tumors in nude mice. TSU cells transfectedwith t-PALP cDNA or mock transfected cells were harvested with 10 mMEDTA-PBS. Transfections were performed as described in Example 51. Onemillion tumor cells suspended in 0.2 ml of DMEM per side was injectedsubcutaneously into 5 week old nude mice (Taconic, Germantown, N.Y.).Five mice were used in each group. The tumor size was measured twice aweek. The mice were sacrificed 4 weeks later and the tumors were removedand weighed and measured. The mean +/− standard error were calculatedand the data were subjected to student t-test for statistical analysis.

In one experiment, the mean tumor volumes (cm³) for nude mice injectedwith mock-transfected tumor cells as compared to nude mice injected witha t-PALP expression vector construct are as follows:

Days Post-injection Mock Tumor Vol. t-PALP Tumor Vol. 3 0.03 0.015 90.075 <0.01 12 0.09 <0.01 16 0.13 <0.01

Thus, animals which were injected with tumor cells transfected with themock control developed tumors increasing in volume over time fromapproximately 0.03 cm³ to approximately 0.13 cm³ from day 3post-injection to day 16 post-injection, respectively. Conversely,animals which were injected with tumor cells transfected with a t-PALPexpression vector developed tumors increasing in volume over time fromapproximately 0.03 cm³ to approximately 0.13 cm³ from day 3post-injection to day 16 post-injection, respectively.

t-PALP may therefore play a regulatory role in tumorigenesis and/oragiogenesis t-PALP, and agonists thereof, may inhibit angiogenesisand/or tumor growth, while antagonists of t-PALP, for example,anti-t-PALP antibodies of the invention, may reduce or block theinhibitory effect of t-PALP with respect to angiogenesis and/ortumorigenesis.

Example 53 Anchorage-Dependenat and Anchorage-Independent Cell GrowthAssay

In this assay, twenty thousand cells were transfected with pcDNA3expression vector alone or with pcDNA3/t-PALP and were seeded in a 24well plate with 1 ml of 10% calf serum-DMEM. Medium was changed everyother day. Transfections were performed as described in Example 51. Thecells were harvested with 1 ml of 10 mM-PBS at day 1, 3, 5, 7, and 9 andcounted with a Coulter counter. In anchorage-dependent growth assay,twenty thousand transfectants in 10% calf serum-DMEM were mixed with0.36% agar and immediately placed on top of 0.6% bottom layer agar inthe same media. Three weeks later, the colonies formed in the soft agarwere quantified using an Omnicon Image Analysis system (Imaging ProductsInternational Inc, Chantilly, Va.).

Example 54 Immunohistochemical Staining of Vessels in Tumors Formed byt-PALP Transfectants

Transfections of pcDNA3 and pcDNA3-t-PALP were performed as described inExample 51 and injected into nude mice as described in Example 52. Thetumors formed by mock transfectants and pcDNA3-t-PALP transfectants wereimmersed into liquid nitrogen immediately after harvested from animals.The frozen sections (10 mm thick) were fixed with aceton and air dry.The sections were stained with 1:500 rabbit anti-factor VIII (a markerof endothelium, DAKO Inc.) and 1:10 Paris (a mouse monoclonal antibodyspecific to chicken endothelium), followed by correspondent ABC kits(containing biotinylated secondary antibody and strepavidin conjugatedperoxidase) and AEC substrate (yield a red color). The number of vesselsin 10 random fields were counted.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

1. An isolated nucleic acid molecule comprising a polynucleotideselected from the group consisting of: (a) a nucleotide sequenceencoding the t-PALP polypeptide having the amino acid sequence atpositions −21 to 242 of SEQ ID NO:2; (b) a nucleotide sequence encodingthe t-PALP polypeptide having the amino acid sequence at positions −20to 242 of SEQ ID NO:2; (c) a nucleotide sequence encoding the maturet-PALP polypeptide having the amino acid sequence at positions 1 to 242in SEQ ID NO:2; (d) a nucleotide sequence encoding the protease domainof the t-PALP polypeptide having the amino acid sequence at positions 64to 242 in SEQ ID NO:2; (e) a nucleotide sequence encoding the completeamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 209023; (f) a nucleotide sequence encoding the complete amino acidsequence excepting the N-terminal methionine encoded by the cDNA clonecontained in ATCC Deposit No. 209023; (g) a nucleotide sequence encodingthe mature t-PALP polypeptide encoded by the cDNA clone contained in theATCC Deposit No. 209023; (h) a nucleotide sequence encoding the proteasedomain of the t-PALP polypeptide encoded by the cDNA clone contained inthe ATCC Deposit No. 209023; and (i) a nucleotide sequence completelycomplementary to any of the complete nucleotide sequences in (a), (b),(c), (d), (e), (f), (g), or (h) above.
 2. A method for making arecombinant vector comprising inserting an isolated nucleic acidmolecule of claim 1 into a vector.
 3. A recombinant vector produced bythe method of claim
 2. 4. A method of making an isolated recombinanthost cell comprising introducing the recombinant vector of claim 3 intoa host cell.
 5. An isolated recombinant host cell produced by the methodof claim
 4. 6. A recombinant method for producing a t-PALP polypeptide,comprising culturing the recombinant host cell of claim 5 underconditions such that said polypeptide is expressed and recovering saidpolypeptide.
 7. The isolated nucleic acid molecule of claim 1 whichcomprises polynucleotide sequence (a).
 8. The isolated nucleic acidmolecule of claim 1 which comprises polynucleotide sequence (b).
 9. Theisolated nucleic acid molecule of claim 1 which comprises polynucleotidesequence (c).
 10. The isolated nucleic acid molecule of claim 1 whichcomprises polynucleotide sequence (d).
 11. The isolated nucleic acidmolecule of claim 1 which comprises polynucleotide sequence (e).
 12. Theisolated nucleic acid molecule of claim 1 which comprises polynucleotidesequence (f).
 13. The isolated nucleic acid molecule of claim 1 whichcomprises polynucleotide sequence (g).
 14. The isolated nucleic acidmolecule of claim 1 which comprises polynucleotide sequence (h).
 15. Theisolated nucleic acid molecule of claim 1 wherein the polynucleotidesequence further comprises a heterologous polynucleotide sequence. 16.The isolated nucleic acid molecule of claim 15 wherein the heterologouspolynucleotide encodes a heterologous polypeptide.
 17. The isolatednucleic acid molecule of claim 16 wherein the heterologous polypeptideis the Fc domain of immunoglobulin, a hexa-histidine peptide, or a FLAGpolypeptide.
 18. A composition comprising the polynucleotide of claim 1and a carrier.
 19. An isolated nucleic acid molecule consisting of anucleotide sequence encoding the kringle domain of the t-PALPpolypeptide consisting of the amino acid sequence at positions 4 to 63in SEQ ID NO:2.
 20. An isolated nucleic acid molecule consisting of anucleotide sequence encoding the kringle domain of the t-PALPpolypeptide encoded by the cDNA clone contained in the ATCC Deposit No.209023.